CYTOTOXIC T LYMPHOCYTES SPECIFIC FOR MUTATED FORMS OF EPIDERMAL GROWTH FACTOR RECEPTOR FOR USE IN TREATING CANCER

Abstract

Compositions, methods, and kits are provided for producing rejuvenated cytotoxic T cells (CTLs) specific for mutated neo-antigen epitopes expressed on cancerous cells, including epidermal growth factor receptor (EGFR) and KRAS neo-antigen epitopes. Antigenspecific CTLs are rejuvenated by reprogramming them into induced pluripotent stem cells (IPSCs) using Yamanaka factors and redifferentiating them back into CTLs while expanding their numbers. After redifferentiation, the IPSC-derived rejuvenated CTLs retain the antigen specificity of the original CTLs from which they were derived, but have the advantage of having longer telomeres and higher proliferative activity than the original CTLs. Pharmaceutical compositions comprising such IPSC-derived rejuvenated CTLs are useful for treating cancers expressing the mutated neo-antigen epitopes recognized by the original CTLs.

Description

BACKGROUND

[0001] Lung cancer is the leading cause of cancer-related deaths worldwide. Overexpression of epidermal growth factor receptor (EGFR) is observed in various malignancies, including lung cancer. EGFR activation induces many intracellular signaling pathways, such as those involving mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), and signal transducer and activator of transcription (STAT) family members (West et al. (2009) J. Thorac. Oncol. 4:s1029-s1039). EGFR activation triggers many intracellular signaling pathways, such as those involving mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), and signal transducer and activator of transcription (STAT), which cause tumor cell proliferation and promote tumor survival (West et al., supra; Jackman et al. (2009) Clin. Cancer Res. 15:5267-5273).

[0002] The EGFR pathway is an appropriate target for cancer therapy. Several agents that block this pathway have been developed and have become the standard of care, first-line treatment for lung cancer patients. EGFR-tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, have demonstrated remarkable clinical activity against non-small cell lung cancer (NSCLC) that harbors activating EGFR mutations. However, patients frequently develop acquired resistance to EGFR-TKI therapy. Replacement of a threonine with a methionine at codon 790 of EGFR (EGFR T790M) is the most common acquired resistance mutation, and is present in .about.50% of cases of TKI resistance (Gao et al. (2016) Expert Rev. Anticancer Ther. 16(4):383-390, Noda et al. (2016) Expert Rev. Respir. Med. 10(5):547-556, van der Wekken et al. (2016) Crit. Rev. Oncol. Hematol. 100:107-116, Villadolid et al. (2015) Transl. Lung Cancer Res. 4(5):576-583, Black et al. (2015) R I Med J (2013) 98(10):25-28). Studies have found when T790M is introduced in vitro into sequences containing wild-type EGFR, an exon 19 deletion-EGFR, or L858R-EGFR, the resulting proteins are significantly more resistant to gefitinib in the constructs containing T790M. These specific mutation sequences are becoming the biosignatures of relapsed cancers (Berman et al. (2016) Transl. Lung Cancer Res. 2016 February; 5(1):138-142). New treatment strategies for NSCLC patients harboring the EGFR T790M mutation are needed.

[0003] Cancer Immunotherapy is a new class of cancer treatment, with unique characteristics that distinguish it from other kinds of cancer therapies. It exploits the fact that cancer cells often have subtly different antigens/molecules that the immune system can detect. Immunotherapy is used to provoke the immune system into attacking tumor cells with these antigens/molecules as targets. Major advantages of cancer immunotherapy over other therapeutic approaches are its high specificity and low toxicity against normal tissues. Adoptive T-cell immunotherapy is a form of cellular immunotherapy that involves transfusion of patients with functional T-cells. This is a potential therapeutic strategy for combating various types of cancer. Recent reports indicate that tumor-reactive T cells recognize various mutated epitopes suggesting that these are potentially immunogenic and, as tumor signatures, might serve as immunotherapeutic targets (Simon et al. (2015) Oncoimmunology 5 (1):e1104448, Hasegawa et al. (2015) PLoS One 10(12)). The effectiveness of adoptive immunotherapy, however, is often hampered by exhaustion of antigen-specific T cells during ex vivo expansion.

SUMMARY

[0004] Compositions, methods, and kits are provided for producing rejuvenated cytotoxic T cells (CTLs) specific for mutated neo-antigen epitopes expressed on cancerous cells, including epidermal growth factor receptor (EGFR) and KRAS neo-antigen epitopes. Antigen-specific CTLs are rejuvenated by reprogramming them into induced pluripotent stem cells (IPSCs) using Yamanaka factors and redifferentiating them back into CTLs while expanding their numbers. After redifferentiation, the IPSC-derived rejuvenated CTLs retain the antigen specificity of the original CTLs from which they were derived, but have the advantage of having longer telomeres and higher proliferative activity than the original CTLs. Pharmaceutical compositions comprising such IPSC-derived rejuvenated CTLs are useful for treating cancers expressing the mutated neo-antigen epitopes recognized by the original CTLs.

[0005] In one aspect, a method of cellular immunotherapy is provided for treating a subject for a cancer expressing a mutated epidermal growth factor receptor (EGFR) or KRAS neo-antigen epitope. In certain embodiments, the method comprises: a) eliciting an antigen-specific cytotoxic T cell response by contacting CTLs with an antigen presenting cell presenting at its surface an immunogenic peptide comprising the mutated EGFR or KRAS neo-antigen epitope in a complex with major histocompatibility complex (MHC); b) isolating CTLs specific for the mutated EGFR or KRAS neo-antigen epitope; c) generating induced pluripotent stem cells (IPSC) from the CTLs specific for the mutated EGFR or KRAS neo-antigen epitope; d) differentiating the IPSCs into rejuvenated CTLs specific for the mutated EGFR or KRAS neo-antigen epitope; and e) administering a therapeutically effective amount of the rejuvenated CTLs specific for the mutated EGFR or KRAS neo-antigen epitope to the subject.

[0006] In certain embodiments, the neo-antigen epitope is a mutated EGFR neo-antigen comprising a mutation selected from the group consisting of a C797S mutation, a T790M mutation, an L858R mutation, and a deletion. In other embodiments, the neo-antigen epitope is a mutated KRAS neo-antigen comprising a mutation selected from the group consisting of a G12D mutation, a G12V mutation, and a G12C mutation.

[0007] In certain embodiments, the immunogenic peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:1-5, or a sequence displaying at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity thereto, wherein the immunogenic peptide comprises the mutated EGFR or KRAS neo-antigen epitope.

[0008] In certain embodiments, the CTLs are contacted with the antigen presenting cell in vivo, ex vivo, or in vitro. The CTLs specific for the mutated EGFR or KRAS neo-antigen epitope may be isolated, for example, from tumor infiltrating lymphocytes or peripheral blood mononuclear cells.

[0009] In certain embodiments, the CTLs are provided in a biological sample. The biological sample may be collected from the subject to be treated or a donor. In certain embodiments, the biological sample is blood, a tumor biopsy, a cancerous tissue sample, or a malignant effusion fluid sample. In one embodiment, the cancerous tissue sample is a lung cancer tissue sample.

[0010] The CTLs may be autologous or allogeneic. In one embodiment, the CTLs are obtained from a donor that is human leukocyte antigen (HLA)-matched with the subject undergoing the cellular immunotherapy.

[0011] In certain embodiments, the antigen presenting cell is a dendritic cell or a macrophage. In other embodiments, the antigen presenting cell is a cancerous cell expressing the mutated epidermal growth factor receptor (EGFR) or KRAS neo-antigen epitope. In further embodiments, an artificial antigen presenting cell is used such as, but not limited to, an MHC multimer, a cellular artificial antigen presenting cell (e.g., fibroblasts or other cells genetically modified to express MHC and other CTL stimulating proteins, or an acellular antigen presenting cell (e.g., biocompatible particle such as a microparticle or nanoparticle carrying CTL stimulating proteins).

[0012] In certain embodiments, the rejuvenated CTLs express CD8.

[0013] In certain embodiments, the rejuvenated CTLs are expanded in vitro before being administered to the subject.

[0014] In certain embodiments, the therapeutically effective amount of the rejuvenated CTLs is provided in a composition. The composition may further comprise a pharmaceutically acceptable excipient. In some embodiments, the composition further comprises an adjuvant. In another embodiment, the composition further comprises an anti-cancer therapeutic agent.

[0015] In certain embodiments, the subject has lung cancer (e.g., non-small cell lung carcinoma).

[0016] In certain embodiments, multiple cycles of treatment are administered to the subject for a time period sufficient to effect at least a partial tumor response, or more preferably, a complete tumor response.

[0017] In certain embodiments, the cancer expresses a major histocompatibility complex (MHC) carrying a peptide comprising the mutated EGFR or KRAS neo-antigen epitope.

[0018] In certain embodiments, the method further comprises introducing a suicide gene into the rejuvenated CTLs. For example, a nucleic acid encoding an inducible caspase-9 may be introduced into the rejuvenated CTLs, wherein induction of expression of the caspase-9 results in apoptosis of the rejuvenated CTLs.

[0019] In another aspect, a method is provided for producing an induced pluripotent stem cell (IPSC)-derived rejuvenated cytotoxic T cell (CTL) specific for a mutated EGFR or KRAS neo-antigen epitope. In certain embodiments, the method comprises: a) obtaining a biological sample comprising cytotoxic T cells (CTLs); b) eliciting an antigen-specific cytotoxic T cell response by contacting cytotoxic T cells (CTLs) with an antigen presenting cell presenting at its surface an immunogenic peptide comprising a mutated EGFR or KRAS neo-antigen epitope in a complex with major histocompatibility complex; c) isolating a CTL specific for the mutated EGFR or KRAS neo-antigen epitope; d) generating an induced pluripotent stem cell (IPSO) from the CTL specific for the mutated EGFR or KRAS neo-antigen epitope; and e) differentiating the IPSO into a rejuvenated CTL specific for the mutated EGFR or KRAS neo-antigen epitope.

[0020] In another aspect, a composition is provided comprising an IPSO-derived rejuvenated CTL specific for a mutated EGFR or KRAS neo-antigen epitope described herein. The composition may further comprise a pharmaceutically acceptable excipient. In another embodiment, the composition further comprises an adjuvant. In a further embodiment, the composition further comprises one or more other anti-cancer therapeutic agents such as, but not limited to, chemotherapeutic agents, immunotherapeutic agents, or biologic agents.

[0021] In another aspect, kits are provided for practicing the methods described herein. In certain embodiments, a kit may comprise IPSO-derived rejuvenated CTLs specific for a mutated EGFR or KRAS neo-antigen epitope or reagents for preparing them. The kit may further comprise instructions for use, including instructions on methods of preparing the IPSC-derived rejuvenated CTLs and/or methods of using them in immunotherapy for treating cancer as described herein.

[0022] In another aspect, an immunogenic peptide is provided comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:1-5, or a sequence displaying at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity thereto, wherein the immunogenic peptide comprises the mutated EGFR or KRAS neo-antigen epitope.

[0023] In another aspect, a composition is provided comprising an immunogenic peptide described herein. The composition may further comprise a pharmaceutically acceptable excipient. In another embodiment, the composition further comprises an adjuvant. In a further embodiment, the composition further comprises one or more other anti-cancer therapeutic agents such as, but not limited to, chemotherapeutic agents, immunotherapeutic agents, or biologic agents.

[0024] In another aspect, an isolated antigen presenting cell is provided comprising a MHC carrying an immunogenic peptide described herein.

[0025] In another aspect, a method of cellular immunotherapy is provided for treating a subject for a cancer expressing a mutated EGFR or KRAS neo-antigen epitope. In certain embodiments, the method comprises: a) obtaining a biological sample comprising cytotoxic T cells (CTLs) from the subject; b) isolating CTLs specific for the mutated EGFR or KRAS neo-antigen epitope from the subject; c) generating induced pluripotent stem cells (IPSCs) from the CTLs specific for the mutated EGFR or KRAS neo-antigen epitope; d) differentiating the IPSCs into rejuvenated CTLs specific for the mutated EGFR or KRAS neo-antigen epitope; and e) administering a therapeutically effective amount of the rejuvenated CTLs specific for the mutated EGFR or KRAS neo-antigen epitope to the subject.

[0026] In another aspect, a method of cellular immunotherapy for treating cancer in a subject is provided, the method comprising eliciting an antigen-specific cytotoxic T cell (CTL) response by administering an immunogenic peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:1-5 to the subject.

[0027] In another aspect, a method of cellular immunotherapy for treating cancer in a subject, the method comprising: a) eliciting an antigen-specific cytotoxic T cell (CTL) response by administering an immunogenic peptide comprising a mutated epidermal growth factor receptor (EGFR) or KRAS neo-antigen epitope to the subject; b) obtaining a biological sample comprising CTLs from the subject; c) isolating CTLs specific for the mutated EGFR or KRAS neo-antigen epitope from the biological sample; d) generating induced pluripotent stem cells (IPSCs) from the CTLs specific for the mutated EGFR or KRAS neo-antigen epitope; e) differentiating the IPSCs into rejuvenated CTLs specific for the mutated EGFR or KRAS neo-antigen epitope; and f) administering a therapeutically effective amount of the rejuvenated CTLs specific for the mutated EGFR or KRAS neo-antigen epitope to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0029] FIG. 1 shows a schematic of the strategy for rejuvenation of antigen-specific T cells using iPSC technology. T-iPS cells were generated from antigen-specific T cells, expanded in vitro, and re-differentiated into antigen-specific T cells.

[0030] FIGS. 2A and 2B show the efficacy of EBV-CTL. FIG. 2A shows light emission monitored as indicator of tumor growth in mice. Around 5 days after tumor inoculation, mice were divided into a control and three experimental groups, and then treated with rejT-iC9-EBV, rejT-NTEBV or original EBVCTL. FIG. 2B shows a graph showing that tumor signals progressively increased in mice without treatment, whereas tumor signals declined in mice treated with original EBVCTL. iC9-iPS derived-EBV CTLs also suppressed tumor signals equal to or greater than the original EBVCTL1.

[0031] FIG. 3A shows in vivo bioluminescent imaging of rejT-iC9-EBV expressing FFluc. NOD-Scid mice inoculated intraperitoneally with EBC-LCL cells and with rejT-iC9-EBV cells received three doses of CID (50 mg) intraperitoneally (n=4). Comparison mice received no CID (n=3). Images of three representative mice from each group are shown. FIG. 3B shows a plot of the bioluminescent T cell signal versus time for the NOD-Scid mice that received the CID and the comparison mice that received no CID.

[0032] FIG. 4 shows EGFR/KRAS neo-antigen candidates.

[0033] FIG. 5 shows results of cytotoxicity assays of CTLs specific for mutated KRAS G12V antigens. The % specific lysis for KRASG12V mutated clones at various effector:target ratios is shown.

[0034] FIGS. 6A and 6B show schematics of the strategy for isolating rare neoantigen-reactive T cells from peripheral blood. FIG. 6A shows isolation of CD8+CTLs, which are mixed with an artificial antigen presenting cell presenting a neo-antigen peptide. FIG. 6B shows FACS isolation and expansion of the neo-antigen-specific CTLs.

DETAILED DESCRIPTION OF EMBODIMENTS

[0035] Compositions, methods, and kits are provided for producing IPSC-derived rejuvenated CTLs specific for mutated neo-antigen epitopes expressed on cancerous cells, including EGFR and KRAS neo-antigen epitopes. Also provided are pharmaceutical compositions comprising such IPSO-derived rejuvenated CTLs and methods of using them for treating cancers expressing the mutated neo-antigen epitopes.

[0036] Before the present compositions, methods, and kits are described, it is to be understood that this invention is not limited to particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0037] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0039] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0040] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.

[0041] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0042] Biological sample. The term "sample" with respect to an individual encompasses any sample comprising CTLs such as blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or cancerous tissue from a surgically resected tumor, malignant effusion fluid samples, or tissue cultures or cells derived or isolated therefrom, and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations, such as cancer cells. The definition also includes samples that have been enriched for particular types of molecules, e.g., nucleic acids, polypeptides, etc.

[0043] The term "biological sample" encompasses a clinical sample. The types of "biological samples" include, but are not limited to: tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, fine needle aspirate, lymph node aspirate, cystic aspirate, a paracentesis sample, a thoracentesis sample, and the like.

[0044] The terms "obtained" or "obtaining" as used herein can also include the physical extraction or isolation of a biological sample (e.g., comprising CTLs) from a subject. Accordingly, a biological sample can be isolated from a subject (and thus "obtained") by the same person or same entity that subsequently produces IPSO-derived rejuvenated CTLs from the CTLS in the sample. When a biological sample is "extracted" or "isolated" from a first party or entity and then transferred (e.g., delivered, mailed, etc.) to a second party, the sample was "obtained" by the first party (and also "isolated" by the first party), and then subsequently "obtained" (but not "isolated") by the second party. Accordingly, in some embodiments, the step of obtaining does not comprise the step of isolating a biological sample.

[0045] In some embodiments, the step of obtaining comprises the step of isolating a biological sample (e.g., a pre-treatment biological sample, a post-treatment biological sample, etc.). Methods and protocols for isolating various biological samples (e.g., a blood sample, a serum sample, a plasma sample, a biopsy sample, an aspirate, etc.) will be known to one of ordinary skill in the art and any convenient method may be used to isolate a biological sample.

[0046] By "immunogenic fragment" is meant a fragment of an immunogen which includes one or more epitopes that can stimulate an immune response, including an antigen-specific cytotoxic T cell response. Immunogenic peptides will typically range between 2 to 15 amino acids in length, including any length within this range such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length. In some embodiments, the immunogenic peptide is at least 2, at least 3, at least 5, at least 7, at least 9, at least 10, at least 11, or at least 12 amino acids in length.

[0047] As used herein, the term "epitope" generally refers to the site on an antigen which is recognized by a T-cell receptor (e.g., on a CTL) and/or an antibody. The epitope may be contained in a short peptide derived from a protein antigen or part of a protein antigen. Several different epitopes may be carried by a single antigenic molecule. The term "epitope" may also include modified amino acids. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0048] An immunogenic fragment can be generated from knowledge of the amino acid and corresponding DNA sequences of an antigen (e.g., EGFR or KRAS), as well as from the nature of particular amino acids (e.g., size, charge, etc.) and the codon dictionary, without undue experimentation. See, e.g., Ivan Roitt, Essential Immunology, 1988; Kendrew, supra; Janis Kuby, Immunology, 1992 e.g., pp. 79-81. Some guidelines in determining whether a protein will stimulate a response, include: Peptide length--typically the peptide is about 8 or 9 amino acids long to fit into a MHC class I complex and about 13-25 amino acids long to fit into a class II MHC complex. Peptides may be longer than these lengths. For example, a longer peptide may be needed if it is partially degraded in cells. The peptide may contain an appropriate anchor motif which will enable it to bind to various class I or class II molecules with high enough specificity to generate an immune response (See Bocchia, M. et al, Specific Binding of Leukemia Oncogene Fusion Protein Pentides to HLA Class I Molecules, Blood 85:2680-2684; Englehard, V H, Structure of peptides associated with class I and class II MHC molecules Ann. Rev. Immunol. 12:181 (1994)).

[0049] The terms "immunogenic" protein or peptide refer to an antigen having an amino acid sequence which elicits an immunological response, including an antigen-specific cytotoxic T cell response. An "immunogenic" protein or peptide, as used herein, includes the full-length sequence of the protein in question, including the precursor and mature forms, analogs thereof, or immunogenic fragments thereof.

[0050] As used herein, the term "CTL epitope" refers generally to those features of a peptide structure which are capable of inducing a CTL response.

[0051] An "immunological response" to an antigen or composition is the development in a subject of a humoral and/or a cellular immune response to an antigen present in the composition of interest. For purposes of the present invention, a "humoral immune response" refers to an immune response mediated by antibody molecules, while a "cellular immune response" is one mediated by T-lymphocytes and/or other white blood cells. One important aspect of cellular immunity involves an antigen-specific response by cytotoxic T cells (CTLs). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. CTLs help induce and promote the destruction or lysis of cancerous cells, infected cells, or damaged cells. Another aspect of cellular immunity involves an antigen-specific response by helper T-cells. Helper T-cells act to help stimulate the function, and focus the activity of, nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface. A "cellular immune response" also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and/or other white blood cells, including those derived from CD4+ and CD8+ T-cells.

[0052] The ability of a particular antigen to stimulate a cell-mediated immunological response may be determined by a number of assays, such as by lymphoproliferation (lymphocyte activation) assays, CTL cytotoxic cell assays (e.g., the interferon-.gamma. (IFN-.gamma.) enzyme-linked immune spot (ELISPOT) assay for measuring IFN-.gamma. secretion from activated CTLs, the calcein release assay for measuring CTL cytotoxicity using calcein to label target cells, intracellular cytokine staining, granzyme B release assay, chromium release assay, JAM test, CD107a mobilization assay, caspase 3 assay, flow cytometric CTL assay) or by assaying for T-lymphocytes specific for the antigen in a sensitized subject. Such assays are well known in the art. See, e.g., Erickson et al., J. Immunol. (1993) 151:4189-4199; Doe et al., Eur. J. Immunol. (1994) 24:2369-2376. Methods of measuring a cell-mediated immune response include measurement of intracellular cytokines or cytokine secretion by T-cell populations, or by measurement of epitope specific T-cells (e.g., by the tetramer technique) (reviewed by Malyguine et al. (2012) Cells 1(2):111-126, Shafer-Weaver et al. (2003) J. Transl. Med. 1(1):14, Takagi et al. (2017) Biochem. Biophys. Res. Commun. 492(1):27-32, Jerome et al. (2003) Apoptosis 8(6):563-571, Hermans et al. (2004) J. Immunol. Methods 1; 285(1):25-40, van Baalen et al. (2008) Cytometry A 73(11):1058-1065, McMichael and O'Callaghan (1998) J. Exp. Med. 187(9)1367-1371, Mcheyzer-Williams et al. (1996) Immunol. Rev. 150:5-21, Lalvani et al. (1997) J. Exp. Med. 186:859-865; herein incorporated by reference.

[0053] The terms "treatment", "treating", "treat" and the like are used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease. The term "treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and/or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease and/or symptom(s), i.e., arresting their development; or (c) relieving the disease symptom(s), i.e., causing regression of the disease and/or symptom(s). Those in need of treatment include those already inflicted (e.g., those with cancer) as well as those in which prevention is desired (e.g., those with increased susceptibility to cancer, those suspected of having cancer, etc.).

[0054] A therapeutic treatment is one in which the subject is inflicted prior to administration and a prophylactic treatment is one in which the subject is not inflicted prior to administration. In some embodiments, the subject has an increased likelihood of becoming inflicted or is suspected of being inflicted prior to treatment. In some embodiments, the subject is suspected of having an increased likelihood of becoming inflicted.

[0055] "Pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.

[0056] "Pharmaceutically acceptable salt" includes, but is not limited to, amino acid salts, salts prepared with inorganic acids, such as chloride, sulfate, phosphate, diphosphate, bromide, and nitrate salts, or salts prepared from the corresponding inorganic acid form of any of the preceding, e.g., hydrochloride, etc., or salts prepared with an organic acid, such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and lactobionate salts. Similarly, salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).

[0057] The terms "tumor," "cancer" and "neoplasia" are used interchangeably and refer to a cell or population of cells whose growth, proliferation or survival is greater than growth, proliferation or survival of a normal counterpart cell, e.g. a cell proliferative, hyperproliferative or differentiative disorder. Typically, the growth is uncontrolled. The term "malignancy" refers to invasion of nearby tissue. The term "metastasis" or a secondary, recurring or recurrent tumor, cancer or neoplasia refers to spread or dissemination of a tumor, cancer or neoplasia to other sites, locations or regions within the subject, in which the sites, locations or regions are distinct from the primary tumor or cancer. Neoplasia, tumors and cancers include benign, malignant, metastatic and non-metastatic types, and include any stage (I, II, III, IV or V) or grade (G1, G2, G3, etc.) of neoplasia, tumor, or cancer, or a neoplasia, tumor, cancer or metastasis that is progressing, worsening, stabilized or in remission. In particular, the terms "tumor," "cancer" and "neoplasia" include carcinomas, such as squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, and small cell carcinoma. These terms include, but are not limited to, lung cancer, including non-small-cell lung carcinoma (e.g., adenocarcinoma, squamous-cell carcinoma and large-cell carcinoma) and small-cell lung carcinoma, breast cancer, prostate cancer, ovarian cancer, testicular cancer, colon cancer, pancreatic cancer, gastric cancer, hepatic cancer, leukemia, lymphoma, adrenal cancer, thyroid cancer, pituitary cancer, renal cancer, brain cancer, skin cancer, head cancer, neck cancer, oral cavity cancer, tongue cancer, and throat cancer.

[0058] By "anti-tumor activity" is intended a reduction in the rate of cell proliferation, and hence a decline in growth rate of an existing tumor or in a tumor that arises during therapy, and/or destruction of existing neoplastic (tumor) cells or newly formed neoplastic cells, and hence a decrease in the overall size of a tumor during therapy. Such activity can be assessed using animal models.

[0059] The term "tumor response" as used herein means a reduction or elimination of all measurable lesions. The criteria for tumor response are based on the WHO Reporting Criteria [WHO Offset Publication, 48-World Health Organization, Geneva, Switzerland, (1979)]. Ideally, all uni- or bidimensionally measurable lesions should be measured at each assessment. When multiple lesions are present in any organ, such measurements may not be possible and, under such circumstances, up to 6 representative lesions should be selected, if available.

[0060] The term "complete response" (CR) as used herein means a complete disappearance of all clinically detectable malignant disease, determined by 2 assessments at least 4 weeks apart.

[0061] The term "partial response" (PR) as used herein means a 50% or greater reduction from baseline in the sum of the products of the longest perpendicular diameters of all measurable disease without progression of evaluable disease and without evidence of any new lesions as determined by at least two consecutive assessments at least four weeks apart. Assessments should show a partial decrease in the size of lytic lesions, recalcifications of lytic lesions, or decreased density of blastic lesions.

[0062] "Substantially purified" generally refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance comprises the majority percent of the sample in which it resides. Typically in a sample, a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.

[0063] The terms "recipient", "individual", "subject", "host", and "patient", are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc. Preferably, the mammal is human.

[0064] The terms "specific binding," "specifically binds," and the like, refer to non-covalent or covalent preferential binding to a molecule relative to other molecules or moieties in a solution or reaction mixture (e.g., specific binding to a particular peptide or epitope relative to other available peptides, such as binding of a CTL T cell receptor to an immunogenic peptide or CTL epitope presented by MHC on an antigen presenting cell). In some embodiments, the affinity of one molecule for another molecule to which it specifically binds is characterized by a K.sub.D (dissociation constant) of 10.sup.-5 M or less (e.g., 10.sup.-6 M or less, 10.sup.-7 M or less, 10.sup.-8 M or less, 10.sup.-9 M or less, 10.sup.-10 M or less, 10.sup.-11 M or less, 10.sup.-12 M or less, 10.sup.-13 M or less, 10.sup.-14 M or less, 10.sup.-15 M or less, or 10.sup.-16 M or less). "Affinity" refers to the strength of binding, increased binding affinity being correlated with a lower K.sub.D.

[0065] The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity. "Antibodies" (Abs) and "immunoglobulins" (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.

[0066] "Antibody fragment", and all grammatical variants thereof, as used herein are defined as a portion of an intact antibody comprising the antigen binding site or variable region of the intact antibody, wherein the portion is free of the constant heavy chain domains (i.e. CH2, CH3, and CH4, depending on antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a "single-chain antibody fragment" or "single chain polypeptide"), including without limitation (1) single-chain Fv (scFv) molecules (2) single chain polypeptides containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety (3) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety and (4) nanobodies comprising single Ig domains from non-human species or other specific single-domain binding modules; and multispecific or multivalent structures formed from antibody fragments. In an antibody fragment comprising one or more heavy chains, the heavy chain(s) can contain any constant domain sequence (e.g. CH1 in the IgG isotype) found in a non-Fc region of an intact antibody, and/or can contain any hinge region sequence found in an intact antibody, and/or can contain a leucine zipper sequence fused to or situated in the hinge region sequence or the constant domain sequence of the heavy chain(s).

Methods

[0067] Compositions, methods, and kits are provided for producing rejuvenated cytotoxic T cells (CTLs) specific for mutated neo-antigen epitopes expressed on cancerous cells, including epidermal growth factor receptor (EGFR) and KRAS neo-antigen epitopes. Antigen-specific CTLs are rejuvenated by reprogramming them into induced pluripotent stem cells (IPSCs) using Yamanaka factors and redifferentiating them back into CTLs while expanding their numbers. After redifferentiation, the IPSC-derived rejuvenated CTLs retain the antigen specificity of the original CTLs from which they were derived, but have the advantage of having longer telomeres and higher proliferative activity than the original CTLs. Pharmaceutical compositions comprising such IPSC-derived rejuvenated CTLs are useful for treating cancers expressing the mutated neo-antigen epitopes recognized by the original CTLs.

[0068] Immunogenic peptides comprising mutated neo-antigen epitopes may be used to elicit antigen-specific CTLs from either healthy individuals or from cancer patients. CTL responses are induced by contacting CTLs with an antigen presenting cell presenting at its surface the immunogenic peptide comprising the mutated neo-antigen epitope in a complex with major histocompatibility complex (MHC). At least one round of stimulation of the CTLs with the immunogenic peptide will be performed to generate a CTL response in order to provide antigen-specific CTLs that recognize a mutated neo-antigen epitope. In some embodiments, multiple rounds of stimulation of the CTLs with the immunogenic peptide may be performed to generate a CTL response capable of producing sufficient antigen-specific CTLs for processing to produce IPSC-derived rejuvenated antigen-specific CTLs for immunotherapy, as described further below. In certain embodiments, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 rounds or more of stimulation of the CTLs with an immunogenic peptide are performed.

[0069] Stimulation of CTLs with an immunogenic peptide (in the presence of an antigen presenting cell) can be performed in vivo, ex vivo, or in vitro. For example, the immunogenic peptide can be administered to a subject to elicit a CTL response followed by collection of a biological sample from the subject comprising antigen-specific CTLs recognizing mutated neo-antigen epitopes. The biological sample may be any sample containing CTLs specific for the mutated neo-antigen epitope such as a blood sample, a sample of peripheral blood mononuclear cell (PBMCs), cancerous tissue in which the CTLS have infiltrated, or a malignant effusion fluid sample. The CTLs can be isolated from a bodily fluid (e.g., blood) or tissue and cultured.

[0070] Alternatively, a biological sample comprising CTLs can be collected from a subject and treated with an immunogenic peptide in the presence of an antigen-presenting cell ex vivo or in vitro to generate antigen-specific CTLs. Examples of suitable antigen presenting cells that can present an immunogenic peptide to CTLs include dendritic cells, macrophages, and activated B cells. Alternately, artificial antigen presenting cells may be used, such as soluble MHC-multimers or cellular or acellular artificial antigen presenting cells. MHC-multimers typically range in size from dimers to octamers (tetramers commonly used) and can be used to display class 1 or class 2 MHC (Hadrup et al. (2009) Nature Methods 6:520-526, Nepom et al. (2003) Antigen 106:1-4, Bakker et al. (2005) Current Opinion in Immunology 17:428-433). Cellular artificial antigen presenting cells may include cells that have been genetically modified to express T-cell co-stimulatory molecules, MHC alleles and/or cytokines. For example, artificial antigen presenting cells have been generated from fibroblasts modified to express HLA molecules, the co-stimulatory signal, B7.1, and the cell adhesion molecules, ICAM-1 and LFA-3 (Latouche et al. (2000) Nature Biotechnology. 18 (4):405-409). Acellular antigen presenting cells comprise biocompatible particles such as microparticles or nanoparticles that carry T cell activating proteins on their surface (Sunshine et al. (2014) Biomaterials. 35 (1): 269-277), Perica et al. (2014) Nanomedicine: Nanotechnology, Biology and Medicine. 10 (1):119-129). For a review of artificial antigen presenting cells, see, e.g., Oelke et al. (2004) Clin. Immunol. 110(3):243-251, Wang et al. (2017) Theranostics. 7(14):3504-3516, Butler et al. (2014) Immunol Rev. 257(1):191-209, Eggermont et al. (2014) Trends Biotechnol. 32(9):4564-4565, Sunshine et al. (2013) Nanomedicine (Lond) 8(7):1173-1189, and Rhodes et al. (2018) Mol. Immunol. 98:13-18; herein incorporated by reference.

[0071] Typically, the immunogenic peptide is at a concentration ranging from about 10 .mu.g/ml to about 40 .mu.g/ml in the biological sample. The immunogenic peptide may be pre-incubated with the antigen presenting cells for periods ranging from 1 to 18 hours prior to stimulation of the CTLs. Culture media may be supplemented with interleukin 2 (IL-2) and interleukin 15 (IL-15) during intervals between stimulations to induce amino acid uptake and protein synthesis in antigen-activated T cells to promote growth and proliferation of antigen-specific CTLs. The antigen-specific CTLs can subsequently be isolated from biological samples, reprogrammed into induced pluripotent stem cells, and redifferentiated into IPSO-derived rejuvenated CTLs that are specific for the mutated neo-antigen epitope recognized by the original CTLs.

[0072] Neoantigens include tumor-associated antigens that are not present in the normal human genome. Immunogenic peptides may include mutated epitopes of neoantigens that are expressed by cancerous cells from any form of cancer including malignant, metastatic and non-metastatic types of cancer, at any stage (I, II, III, IV or V) or grade (G1, G2, G3, etc.), including carcinomas, such as squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, and small cell carcinoma. In certain embodiments, immunogenic peptides include mutated epitopes of neoantigens expressed by lung cancer, including non-small-cell lung carcinoma (e.g., adenocarcinoma, squamous-cell carcinoma and large-cell carcinoma) and small-cell lung carcinoma, breast cancer, prostate cancer, ovarian cancer, testicular cancer, colon cancer, pancreatic cancer, gastric cancer, hepatic cancer, leukemia, lymphoma, adrenal cancer, thyroid cancer, pituitary cancer, renal cancer, brain cancer, skin cancer, head cancer, neck cancer, oral cavity cancer, tongue cancer, and throat cancer.

[0073] An immunogenic peptide comprising a mutated neoantigen CTL epitope can be designed based on knowledge of the amino acid sequence of the mutated neoantigen of interest (e.g., expressed by a cancer in a patient undergoing treatment). Typically, the immunogenic peptide will range in size from 8-12 amino acids in length (i.e., in order to fit into the MHC class I complex for presentation to CTLs), though immunogenic peptides may be longer, particularly if the immunogenic peptide is degraded in cells or the biological sample. The immunogenic peptide may further contain an appropriate anchor motif which will enable it to bind to various MHC class I or class II molecules with high enough specificity to generate an immune response (See Bocchia, M. et al, Specific Binding of Leukemia Oncogene Fusion Protein Pentides to HLA Class I Molecules, Blood 85:2680-2684; Englehard, V H, Structure of peptides associated with class I and class II MHC molecules Ann. Rev. Immunol. 12:181 (1994)). The sequence of a neoantigen of interest can be compared to published structures of peptides associated with MHC molecules. Representative MHC binding peptides can be found in a number of databases including, the MHCBN, JenPep, MHCPEP, and SYFPEITHI databases. In addition, epitope prediction software can be used for prediction of MHC binding peptides and CTL epitopes for various MHC alleles. For example, nHLAPred (crdd.osdd.net/raghava/nhlapred/) uses artificial neural networks (ANNs) and quantitative matrices (QM) for prediction of MHC binding peptides and CTL epitopes for various MHC alleles. ProPredl (crdd.osdd.net/raghava/propredl/) identifies MHC Class-I binding regions in antigens for MHC Class-I alleles. BIMAS (bimas.cit.nih.gov/molbio/hla_bind/, Lefranc et al. (2003) Leukemia 17:260-266) predicts MHC-binding peptides based on their predicted half-time of dissociation from MHC class I alleles. RANKPEP ranks peptides based on their sequences according to their similarity to peptides known to bind to a given MHC molecule. PREDEP (margalit.huji.ac.il/Teppred/mhc-bind/) is a structure-based algorithm for prediction of MHC class I epitopes. MMBPred (crdd.osdd.net/raghava/mmbpred/) predicts mutated MHC class-I binding peptides in antigenic proteins for MHC class I alleles.

[0074] In certain embodiments, the immunogenic peptide comprises a mutated EGFR neo-antigen epitope comprising a mutation selected from the group consisting of a C797S mutation, a T790M mutation, an L858R mutation, and a deletion. In other embodiments, the immunogenic peptide comprises a mutated KRAS neo-antigen epitope comprising a mutation selected from the group consisting of a G12D mutation, a G12V mutation, and a G12C mutation. In certain embodiments, the immunogenic peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:1-5, or a sequence displaying at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity thereto, wherein the immunogenic peptide comprises a mutated EGFR or KRAS neo-antigen epitope.

[0075] The ability of a particular immunogenic peptide to stimulate a CTL cell-mediated immunological response may be determined by a number of assays, such as by lymphoproliferation (lymphocyte activation) assays, CTL cytotoxic cell assays (e.g., the interferon-.gamma. (IFN-.gamma.) enzyme-linked immune spot (ELISPOT) assay for measuring IFN-.gamma. secretion from activated CTLs, the calcein release assay for measuring CTL cytotoxicity using calcein to label target cells, intracellular cytokine staining, granzyme B release assay, chromium release assay, JAM test, CD107a mobilization assay, caspase 3 assay, flow cytometric CTL assay) or by assaying for CTLs specific for the antigen in a sensitized subject. Such assays are well known in the art. See, e.g., Erickson et al., J. Immunol. (1993) 151:4189-4199; Doe et al., Eur. J. Immunol. (1994) 24:2369-2376. Methods of measuring a cell-mediated immune response include measurement of intracellular cytokines or cytokine secretion by T-cell populations, or by measurement of epitope specific T-cells (e.g., by the tetramer technique) (reviewed by Malyguine et al. (2012) Cells 1(2):111-126, Shafer-Weaver et al. (2003) J. Transl. Med. 1(1):14, Takagi et al. (2017) Biochem Biophys Res Commun. 492(1):27-32, Jerome et al. (2003) Apoptosis 8(6):563-571, Hermans et al. (2004) J. Immunol. Methods 1; 285(1):25-40, van Baalen et al. (2008) Cytometry A 73(11):1058-1065, McMichael and O'Callaghan (1998) J. Exp. Med. 187(9)1367-1371, Mcheyzer-Williams et al. (1996) Immunol. Rev. 150:5-21, Lalvani et al. (1997) J. Exp. Med. 186:859-865; herein incorporated by reference.

[0076] The antigen-specific CTLs can optionally be purified before or after reprogramming and redifferentiation by any method known in the art, including, but not limited to, density gradient centrifugation (e.g., Ficoll Hypaque, percoll, iodoxanol and sodium metrizoate), immunoselection (positive selection or negative selection for surface markers) with immunomagnetic beads or immunoaffinity columns, or fluorescence-activated cell sorting (FACS). See, e.g., Cytotoxic T-Cells, Methods and Protocols (E. Ranieri, ed., Humana Press, 2014), Thiery et al. (2010) Curr. Protoc. Cell Biol. Chapter 3:Unit 3.37, and Oelke et al. (2000) Clin. Cancer Res. 6(5):1997-2005; herein incorporated by reference.

Reprogramming Antigen-Specific Cytotoxic T Cells

[0077] Rejuvenated antigen-specific CTLs and be generated by reprogramming the original CTLs obtained from a subject into pluripotent stem cells followed by redifferentiation. CTLs are induced into forming pluripotent stem cells, for example, by treating them with reprograming factors such as Yamanaka factors, including but not limited to, OCT3, OCT4, SOX2, KLF4, c-MYC, NANOG, and LIN28 (see, e.g., Nishimura et al. (2013) Cell Stem Cell 12:114-126, Takayama et al. (2010) J. Exp. Med. 207:2817-2830, and U.S. Pat. No. 9,206,394; herein incorporated by reference in their entireties). After in vitro expansion, the CTL-derived IPSCs can be redifferentiated into hematopoietic cells by culturing the IPSCs in the presence of VEGF, SCF, and FLT-3L. The hematopoietic cells can subsequently be redifferentiated into CTLs expressing a desired T cell receptor by culturing in the presence of FLT-3L and IL-7. After such redifferentiation, the CTLs are now rejuvenated (i.e., IPSC-derived rejuvenated CTLs have longer telomeres and higher proliferative activity than the original CTLs while retaining the specificity for neo-antigen epitopes recognized by the original CTLs). For redifferentiation protocols, see, e.g., Nishimura et al., supra; Takayama et al., supra; Timmermans et al. (2009) J. Immunol. 182:6879-6888, and Ikawa et al. (2010) Science 329:93-96; herein incorporated by reference in their entireties.

[0078] Methods for "introducing a cell reprogramming factor into CTLs are not limited in particular, and known procedures can be selected and used as appropriate. For example, when a cell reprogramming factor as described above is introduced into CTLs of the above-mentioned type in the form of proteins, such methods include ones using protein introducing reagents, fusion proteins with protein transfer domains (PTDs), electroporation, and microinjection. When a cell reprogramming factor as described above is introduced into CTLs of the above-mentioned type in the form of nucleic acids encoding the cell reprogramming factor, a nucleic acid(s), such as cDNA(s), encoding the cell reprogramming factor can be inserted in an appropriate expression vector comprising a promoter that functions in CTLs, which then can be introduced into CTLs by procedures such as infection, lipofection, liposomes, electroporation, calcium phosphate coprecipitation, DEAE-dextran, microinjection, and electroporation.

[0079] Examples of an "expression vector" include viral vectors, such as lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, and herpes viruses; and expression plasmids for animal cells. For example, retroviral or Sendai virus (SeV) vectors are commonly used to introduce a nucleic acid(s) encoding a cell reprogramming factor as described above into CTLs.

[0080] In addition, a suicide gene may be introduced into the IPSO-derived rejuvenated CTLs, for example, to improve their safety by allowing their destruction at will. Suicide genes can be used to selectively kill cells by inducing apoptosis or converting a nontoxic drug to a toxic compound in the CTLs. Examples include suicide genes encoding caspases, thymidine kinases, cytosine deaminases, intracellular antibodies, telomerases, and DNases. See, e.g., Jones et al. (2014) Front. Pharmacol. 5:254, Mitsui et al. (2017) Mol. Ther. Methods Clin. Dev. 5:51-58, Greco et al. (2015) Front. Pharmacol. 6:95; herein incorporated by reference. In some cases, the suicide gene is expressed from an inducible promoter to provide a "safety switch" (i.e., kill cells by inducing the suicide gene). For example, an inducible caspase-9 suicide gene system can be incorporated into IPSO-derived rejuvenated CTLs as a "safety switch" (see, e.g., Straathof et al. (2005) Blood 105(11):4247-4254; Thomis et al. (2001) Blood 97(5):1249-1257; Tey et al. (2007) Biol. Blood Marrow Transplant. 13(8):913-24; herein incorporated by reference.). In some embodiments, a suicide gene is selected that expresses a human protein to minimize immune reactions in human patients treated with the CTLs.

Pharmaceutical Compositions and Cellular Immunotherapy with Rejuvenated CTLs

[0081] Pharmaceutical compositions can be prepared by formulating the IPSO-derived rejuvenated CTLs produced by the methods described herein into dosage forms by known pharmaceutical methods. For example, a pharmaceutical composition comprising IPSC-derived rejuvenated CTLs can be formulated for parenteral administration, as capsules, liquids, film-coated preparations, suspensions, emulsions, and injections (such as venous injections, drip injections, and the like).

[0082] In formulation into these dosage forms, the IPSO-derived rejuvenated CTLs can be combined as appropriate, with pharmaceutically acceptable carriers or media, in particular, sterile water and physiological saline, vegetable oils, resolvents, bases, emulsifiers, suspending agents, surfactants, stabilizers, vehicles, antiseptics, binders, diluents, tonicity agents, soothing agents, bulking agents, disintegrants, buffering agents, coating agents, lubricants, coloring agents, solution adjuvants, or other additives. The IPSO-derived rejuvenated CTLs may be also used in combination with known pharmaceutical compositions, immunostimulants, anti-cancer agents, or other therapeutic agents.

[0083] In some embodiments, the pharmaceutical composition comprising the IPSO-derived rejuvenated CTLs is a sustained-release formulation, or a formulation that is administered using a sustained-release device. Such devices are well known in the art, and include, for example, transdermal patches, and miniature implantable pumps that can provide for delivery of the IPSO-derived rejuvenated CTLs over time in a continuous, steady-state fashion at a variety of doses to achieve a sustained-release effect with a non-sustained-release pharmaceutical composition.

[0084] Usually, but not always, the subject who receives the IPSO-derived rejuvenated CTLs (i.e., the recipient) is also the subject from whom the original CTLs (i.e., before rejuvenation) are harvested or obtained, which provides the advantage that the cells are autologous. However, CTLs can be obtained from another subject (i.e., donor), a culture of cells from a donor, or from established cell culture lines and rejuvenated according to the methods described herein. CTLs may be obtained from the same or a different species than the subject to be treated, but preferably are of the same species, and more preferably of the same immunological profile as the subject. Such cells can be obtained, for example, from a biological sample comprising CTLs from a close relative or matched donor, then reprogrammed into induced pluripotent stem cells (IPSCs) using Yamanaka factors and redifferentiated into the IPSO-derived rejuvenated CTLs and administered to a subject in need of treatment for cancer.

[0085] In certain embodiments, the IPSC-derived rejuvenated CTLs administered to a subject are autologous or allogeneic. The patients or subjects who donate or receive the CTLs are typically mammalian, and usually human. However, this need not always be the case, as veterinary applications are also contemplated.

[0086] At least one therapeutically effective dose of the IPSC-derived rejuvenated CTLs will be administered. By "therapeutically effective dose or amount" of the IPSC-derived rejuvenated CTLs is intended an amount that when administered brings about a positive therapeutic response with respect to treatment of an individual for cancer. Of particular interest is an amount of the IPSC-derived rejuvenated CTLs that provides an anti-tumor effect, as defined herein. By "positive therapeutic response" is intended the individual undergoing the treatment according to the invention exhibits an improvement in one or more symptoms of the cancer for which the individual is undergoing therapy.

[0087] Thus, for example, a "positive therapeutic response" would be an improvement in the disease in association with the therapy, and/or an improvement in one or more symptoms of the disease in association with the therapy. Therefore, for example, a positive therapeutic response would refer to one or more of the following improvements in the disease: (1) reduction in tumor size; (2) reduction in the number of cancer cells; (3) inhibition (i.e., slowing to some extent, preferably halting) of tumor growth; (4) inhibition (i.e., slowing to some extent, preferably halting) of cancer cell infiltration into peripheral organs; (5) inhibition (i.e., slowing to some extent, preferably halting) of tumor metastasis; and (6) some extent of relief from one or more symptoms associated with the cancer. Such therapeutic responses may be further characterized as to degree of improvement. Thus, for example, an improvement may be characterized as a complete response. By "complete response" is documentation of the disappearance of all symptoms and signs of all measurable or evaluable disease confirmed by physical examination, laboratory, nuclear and radiographic studies (i.e., CT (computer tomography) and/or MRI (magnetic resonance imaging)), and other non-invasive procedures repeated for all initial abnormalities or sites positive at the time of entry into the study. Alternatively, an improvement in the disease may be categorized as being a partial response. By "partial response" is intended a reduction of greater than 50% in the sum of the products of the perpendicular diameters of all measurable lesions when compared with pretreatment measurements (for patients with evaluable response only, partial response does not apply).

[0088] The pharmaceutical compositions comprising the IPSC-derived rejuvenated CTLs may be administered using any route of administration in accordance with any medically acceptable method known in the art. Suitable routes of administration include parenteral administration, such as intravenous (IV), intraarterial, infusion, subcutaneous (SC), intraperitoneal (IP), intramuscular (IM), pulmonary, nasal, topical, or transdermal. In some embodiments, the pharmaceutical composition comprising the IPSC-derived rejuvenated CTLs is administered locally to the site of a tumor or cancerous cells.

[0089] Factors influencing the respective amount of the various compositions to be administered include, but are not limited to, the mode of administration, the frequency of administration, the particular type of cancer undergoing therapy, the severity of the disease, the history of the disease, whether the individual is undergoing concurrent therapy with another therapeutic agent, and the age, height, weight, health, and physical condition of the individual undergoing therapy. Generally, a higher dosage is preferred with increasing weight of the subject undergoing therapy.

[0090] In certain embodiments, multiple therapeutically effective doses of the IPSC-derived rejuvenated CTLs will be administered for a time period sufficient to effect at least a partial tumor response and more preferably a complete tumor response. Where a subject undergoing immunotherapy exhibits a partial response, or a relapse following a prolonged period of remission, subsequent courses of immunotherapy may be needed to achieve complete remission of the disease. Thus, subsequent to a period of time off from a first treatment period, a subject may receive one or more additional treatment periods comprising immunotherapy with IPSO-derived rejuvenated CTLs. Such a period of time off between treatment periods is referred to herein as a time period of discontinuance. It is recognized that the length of the time period of discontinuance is dependent upon the degree of tumor response (i.e., complete versus partial) achieved with any prior treatment periods of immunotherapy with the IPSC-derived rejuvenated CTLs or other therapeutic agents.

Kits

[0091] Also provided are kits for practicing the methods described herein. In certain embodiments, the kit comprises IPSO-derived rejuvenated CTLs specific for a mutated EGFR or KRAS neo-antigen epitope or reagents for preparing them. For example, the kit may comprise an immunogenic peptide comprising a mutated EGFR or KRAS neo-antigen epitope, an antigen presenting cell (e.g., dendritic cell, macrophage, or cellular or acellular artificial antigen presenting cell (e.g., MHC multimer)), agents for isolating CTLs (e.g., immunomagnetic beads or immunoaffinity columns), reprograming factors (e.g., Yamanaka factors such as OCT3, OCT4, SOX2, KLF4, c-MYC, NANOG, and LIN28), redifferentiation factors (e.g., VEGF, SCF, FLT-3L, and IL-7), and culture media. Alternatively, the kit may comprise IPSO-derived rejuvenated CTLs specific for a mutated EGFR or KRAS neo-antigen epitope in a pharmaceutical composition suitable for use in treatment.

[0092] In certain embodiments, the kit comprises an immunogenic peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:1-5, or a sequence displaying at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity thereto, wherein the immunogenic peptide comprises a mutated EGFR or KRAS neo-antigen epitope.

[0093] Kits may comprise one or more containers of the compositions described herein. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, including glass or plastic. A container may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The kit can further comprise a container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can also contain other materials useful to the end-user, including other pharmaceutically acceptable formulating solutions such as buffers, diluents, filters, needles, and syringes or other delivery device. The kit may also provide a delivery device pre-filled with the IPSC-derived rejuvenated CTLs.

[0094] In addition to the above components, the subject kits may further include (in certain embodiments) instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like. Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), DVD, Blu-ray, flash drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site.

Utility

[0095] The IPSO-derived rejuvenated CTLs, produced by the methods described herein, are useful in cellular immunotherapy for treating cancer, particularly cancers expressing mutated EGFR or KRAS neo-antigen epitopes.

[0096] The term "cancer", as used herein, refers to a variety of conditions caused by the abnormal, uncontrolled growth of cells. Cells capable of causing cancer, referred to as "cancer cells", possess characteristic properties such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and/or certain typical morphological features. A cancer can be detected in any of a number of ways, including, but not limited to, detecting the presence of a tumor or tumors (e.g., by clinical or radiological means), examining cells within a tumor or from another biological sample (e.g., from a tissue biopsy), measuring blood markers indicative of cancer, and detecting a genotype indicative of a cancer. However, a negative result in one or more of the above detection methods does not necessarily indicate the absence of cancer, e.g., a patient who has exhibited a complete response to a cancer treatment may still have a cancer, as evidenced by a subsequent relapse.

[0097] The term "cancer" as used herein includes carcinomas, (e.g., carcinoma in situ, invasive carcinoma, metastatic carcinoma) and pre-malignant conditions, i.e. neomorphic changes independent of their histological origin. The term "cancer" is not limited to any stage, grade, histomorphological feature, invasiveness, aggressiveness or malignancy of an affected tissue or cell aggregation. In particular stage 0 cancer, stage I cancer, stage II cancer, stage III cancer, stage IV cancer, grade I cancer, grade II cancer, grade III cancer, malignant cancer and primary carcinomas are included.

[0098] Cancers and cancer cells that can be treated with IPSO-derived rejuvenated CTLs include, but are not limited to, hematological cancers, including leukemia, lymphoma and myeloma, and solid cancers, including for example tumors of the brain (glioblastomas, medulloblastoma, astrocytoma, oligodendroglioma, ependymomas), carcinomas, e.g. carcinoma of the lung, liver, thyroid, bone, adrenal, spleen, kidney, lymph node, small intestine, pancreas, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, and esophagus; sarcomas, melanomas; myelomas; etc.

[0099] In particular, lung cancer may be responsive to treatment with IPSO-derived rejuvenated CTLs specific for mutated EGFR or KRAS neo-antigen epitopes including, without limitation, non-small-cell lung carcinoma (e.g., adenocarcinoma, squamous-cell carcinoma and large-cell carcinoma) and small-cell lung cancer. In an embodiment, the lung cancer is non-small cell lung carcinoma (NSCLC). In certain embodiments, the NSCLC comprises a mutated EGFR neo-antigen comprising a mutation selected from the group consisting of a C797S mutation, a T790M mutation, an L858R mutation, and a deletion. In other embodiments, the NSCLC comprises a mutated KRAS neo-antigen comprising a mutation selected from the group consisting of a G12D mutation, a G12V mutation, and a G12C mutation.

[0100] It will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.

EXPERIMENTAL

[0101] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[0102] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0103] The present invention has been described in terms of particular embodiments found or proposed by the present inventor to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. For example, due to codon redundancy, changes can be made in the underlying DNA sequence without affecting the protein sequence. Moreover, due to biological functional equivalency considerations, changes can be made in protein structure without affecting the biological action in kind or amount. All such modifications are intended to be included within the scope of the appended claims.

Example 1

Introduction

[0104] The discovery of iPSC technology created promising new avenues for treatment.sup.2,3,4,5,6,7. Human iPSCs are a potential source of stem cells for transfusion therapies. The development of widely varying reprogramming methods has enabled us nowadays to obtain iPSCs from even a small number of antigen-specific T cells of patient origin. As these T cell-derived iPSCs (T-iPSCs) carry TCR gene rearrangements in their genomic DNA, they are likely useful for producing antigen-specific T cells and for studying T cell development. T cell immunotherapy is potentially an effective therapeutic strategy against many types of cancers and viral infections. If antigen-specific T cells tailored against diseases and for patients can be easily obtained, T cell immunotherapy should become a popular choice of therapy. However, use of T cell immunotherapy is restricted by HLA alleles. In addition, expansion of CTLs ex vivo has hitherto resulted in production of CTLs with short telomeres and an "exhausted" phenotype. Our laboratory developed an in vitro way to reprogram antigen-specific CTLs to T-iPSCs for expansion and then to guide them sequentially to yield T-lineage cells and mature CD8 single-positive T cells. These CD8+ T cells generated in vitro display antigen-specific cytotoxicity and enhanced proliferative capacity with longer telomeres. Since these T cells originate from a patient's own CTLs, HLA restriction is not an issue. This novel technique thus provides approaches to generate rejuvenated antigen-specific T cells in unlimited numbers. This discovery should resolve issues related to T cell adoptive immunotherapy both qualitatively and quantitatively.

[0105] T lymphocytes play a central role in acquired immunity and control systemic immunity against internal and external pathogens. CTLs and helper lymphocytes are important components of the immune system in the fight against cancers.sup.9,10. These T lymphocytes start to exert their proliferative functions when, via TCRs, they recognize antigens in an HLA-restricted and antigen-specific manner. Adoptive T cell immunotherapy that exploits these features is evolving as a technology with the potential of providing ways safely and effectively to target pathogens for destruction. The greatest advantages of adoptive T cell immunotherapy lie in specific recognition of target cells and in long-term immunological surveillance by long-lived native T lymphocytes.

[0106] In fact, successful treatment of cancers with allogeneic T lymphocytes is a direct proof that human T-cell immunity has the potential to eradicate cancers. However, the effectiveness of adoptive T-cell immunotherapy is often hampered by insufficient recognition of cancer antigens (principally self-antigens), on cancer cells. It is also true that continuous exposure to cancer/self-antigens drives T lymphocytes into a highly exhausted state, with loss of potential for long-term survival, proliferation, and killing functions.sup.10. Several researchers have endeavored to develop clinical protocols for expanding antigen-responding T cells, i.e., tumor-infiltrating lymphocytes, from the few native T cell pools remaining in the patient. Highly expanded T cells in such protocols have not proved fully effective so far because of functional losses incurred during ex vivo manipulation. To overcome these obstacles in cell-based immunotherapy, we endeavored to generate iPS cells from a single T cell of a cancer patient. iPSCs have a capacity for unlimited self-renewal while maintaining pluripotency.sup.9. Unlike other somatic cell-derived iPS cells, TiPSCs have properly rearranged TCRs even after having undergone nuclear reprogramming.

Example 2

Rejuvenation of Antigen-Specific Cytotoxic T Cells

[0107] We have recently developed a novel system in which antigen-specific cytotoxic T cells (CTLs) can be rejuvenated by reprogramming them into induced pluripotent stem cells (iPSCs) and redifferentiating them while expanding their numbers, yielding abundant rejuvenated T cells (rejT cells) (Generation of rejuvenated antigen-specific T cells by reprogramming to pluripotency and redifferentiation. 2013 Jan. 3; 12(1):114-26. Cell Stem Cell). This unique technique has been deployed in vivo with a safeguard system as a model of iPSC-derived, rejCTL therapy (A safeguard system for induced pluripotent stem cell-derived rejuvenated T cell therapy. 2015 Oct. 13; 5(4):597-608. Stem Cell Report). The purpose of this innovative method, the first exploration of the concept of a "kill switch", is to ensure that using iPSC-based therapy in humans is safe.

[0108] Adoptive T-cell immunotherapy has shown promise in treating melanoma and other cancers; however, cytotoxic T-cells can become exhausted, with loss of efficacy during ex vivo expansion. To overcome this obstacle, we have developed a novel system in which antigen-specific T cells are reprogrammed to pluripotent stem cells (T-iPSCs) using Yamanaka factors. After expansion, these T-iPSCs are redifferentiated to functional T cells. They retain their original antigen specificity. These newly redifferentiated T cells display a naive T cell phenotype, with longer telomeres and higher proliferative activity. These iPSCs generated from human T lymphocytes (T-iPSCs) retain their T-cell receptor (TCR) specificity in the genome as encoded by rearranged TCR alpha and beta genes.sup.16,17,18. Because T-iPS cells have unlimited self-renewal capacity, they can be expanded ex vivo. When these T-iPS cells are re-differentiated into CD3+TCR, they are newly generated T cells with original antigen specificity but longer telomeres.sup.11,8. We have demonstrated killing activity and specificity of these "rejuvenated" T cells in vivo. In fact, rejCTL cells show more robust biological activity than the original T cells.

[0109] This unique approach differs from chimeric antigen receptor T-cell (CART) immunotherapy. It uses T cell receptors to recognize non-self-antigens/epitopes expressed inside tumor cells; therefore, this form of immunotherapy using rejCTL cells is restricted by the HLA type. The advantages include that 1) once a iPS cell line is established, T-iPSCs can be generated indefinitely from them, producing young and active T cells without limitation; 2) T-iPSCs can be frozen for future use in patients with the same HLA type and mutation profile; 3) a safeguard system using inducible caspase 9 (iCas9) can be activated to eliminate all T-iPSC-derived cells in case of immunotherapy-associated complications; 4) it offers the opportunity to search for yet unknown cancer epitopes by searching T-iPSC libraries generated from tumor-infiltrating T cells. Application of this technology in lung cancer treatment will open a novel avenue for translational cancer therapies.

[0110] T cells are superior to antibodies in that they can recognize antigenic epitopes inside target cells, epitopes that are presented utilizing the MHC-based system. A disadvantage is perhaps that their ability to recognize antigens is restricted by the MHC allotype. This MHC-based restriction has been an issue in T cell immunotherapy. However, the issue has been addressed by the recent development of iPSC technology enabling the generation of iPSCs from a patient's own T cells. Utilizing this technology, we have developed a system to rejuvenate exhausted CTLs through reprogramming and redifferentiation. This should permit novel adoptive immunotherapies for cancer and viral infections.

[0111] In addition, a safeguard system using iCas9 engineered iPSCs can be applied to any first-in-man study using iPSC- or embryonic stem cell (ESC)-derived cells. Patients' tumor infiltrating T cells can be used to make T-iPSC libraries, followed by clonal redifferentiation of CTLs to search for novel cancer epitopes. This approach may serve to identify yet unknown targets for future use in cancer immunotherapy.

Example 3

Antigen-Specific Cytotoxic T Cells Targeting Mutated Epitopes of EGFR

Introduction

[0112] Mutation-associated epitopes of the receptor tyrosine kinase, EGFR, are commonly present in lung and other forms of cancer. In lung cancer, mutations that activate epidermal growth factor receptor (EGFR) are often found in exons 18 to 21 of EGFR, the portion of the gene that encodes the tyrosine kinase domain of EGFR protein. Exon 19 deletions and exon 21 point mutations account for around 90% of all EGFR mutations in advanced NSCLC. EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, show therapeutic efficacy against NSCLC when such EGFR mutations are present. However, patients frequently develop resistance to EGFR-TKIs with a secondary mutation, a threonine to methionine change at codon 790 of EGFR (EGFR T790M). This is the major mechanism of EGFR-TKI resistance. It causes cancer relapse. Secondary mutations that occur in EGFR are the main mechanism of resistance to tyrosine kinase inhibitors (TKI) active against primarily mutations of EGFR. Mutations in EGFR are often found in cancers arising outside the lung, such as in the pancreas or breast. Without being bound by theory, we propose that T-cell based immunotherapy will not only work effectively against lung cancer but also other solid tumors having the same EGFR mutations. In addition, this approach can also be applied to mutations in other genes associated with cancer (MET, IGF-1R, etc.). Collectively, development of rejuvenated T-iPSCs for lung cancer immunotherapy may have a broad impact on future iPSC-mediated clinical therapy of cancer.

Peptide Prediction and Synthesis, Based on HLA Alleles, of the Peptides Representing EGFR Mutations and Selection of Those with Highest Affinity by Peptide Binding Assay.

[0113] We use the epitope prediction software, BIMAS (bimas.cit.nih.gov/molbio/hla_bind/), to predict peptides that can bind to various HLA alleles.sup.10,11,12.

Generation of Rejuvenated, iC9-Implemented CTLs In Vitro.

[0114] The entire process of generating rejCTLs can be divided into the following steps: A) Induction of CTLs specific to EFGR epitopes carrying mutations (e.g., T790M, deletion and L858R) that were selected by the in-silicon approach. Peripheral-blood mononuclear cells (PBMNCs) contain some mutant EGFR-specific T cells. Because mutant EGFR-specific T cells also infiltrate into primary lung cancer tissue, they can be isolated from such tissue and cultured. Selected peptides are used to treat the original T cells. Peptide-specific responsive T cells are selected using tetramer and FACS isolation. B) Generation of T-iPSCs from EGFR mutation-specific CTLs and implementation of a iC9 based safeguard system.sup.1. The CTLs generated in A) are reprogrammed into T-iPSCs using Sendai virus. The iC9 system is implemented in the T-iPSCs. C) Redifferentiation of CTLs from T-iPSCs. After in vitro expansion, T-iPSCs are redifferentiated into abundant rejCTLs carrying inducible iC9. Antigen specificity, killing activity, and proliferation activity are assayed in vitro.

Evaluation of the Therapeutic Efficacy of the Series of rejCTLs Recognizing Different Mutant EGFR Epitopes.

[0115] Infusion of patient rejCTLs into tumor-grafted mice and evaluation of tumor regression (speed and completeness). The rejCTLs are injected intraperitoneally into patient tumor-grafted mice. Control CTLs, as originally isolated, are injected into xenografted mice to permit comparisons. Tumor sizes are monitored after rejCTL cell injection.sup.1. Timely imaging is used to document changes in the tumor in vivo. The most efficient epitopes for EGFR mutant NSCLCs will demonstrate changes in tumor size in vivo.

Example 4

Antigen-Specific Cytotoxic T Cells Targeting NSCLC Mutated Tumor Epitopes

[0116] The immunogenicity of EGFR and Ras mutations found in NSCLC in association with various HLA alleles are evaluated, and CTLs specific to the mutation sequences are generated. By reprogramming and redifferentiating these NSCLC-specific CTLs, rejCTLs are obtained. In the presence of certain human leukocyte antigen (HLA) alleles, a mutated protein such as that in EGFR T790M-harboring cancer cells is presented as a tumor-specific antigen and is targeted by activated immune cells. We screen various EGFR and KRas mutations (Table 1) and assess their binding affinity to various HLA alleles; the immunogenicity of the mutation-derived peptide sequences with particular HLA molecules is tested in vitro, using transporter associated with antigen processing (TAP)-deficient cell lines.

TABLE-US-00001 TABLE 1 EGFR/KRAS Neo-Antigen Candidates Driver Peptide Gene Mutation ID Peptide Sequence EGFR C797S CS9.3 QLMPFGSLL (SEQ ID NO: 1) C797S CS11.6 LMPFGSLLDYV (SEQ ID NO: 2) KRAS G12D GD10.3 KLVVVGADGV (SEQ ID NO: 3) G12V GV10.3 KLVVVGAVGV (SEQ ID NO: 4) G12C GC10.3 KLVVVGACGV (SEQ ID NO: 5)

TABLE-US-00002 TABLE 2 CDR1 CDR2 CDR3 Frequency TCRalpha DSVNN IPSGT AVDNYGQNFV 770758 Donor Y (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 130) (CS9.3) DSVNN IPSGT AVGNYGQNFV 2095 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 131) DSVNN IPSGT AVDSYGQNFV 1969 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 132) DSVNN IPSGT AADNYGQNFV 1942 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 133) DSVNN IPSGT AVDDYGQNFV 1404 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 134) SSVSVY YLSGSTLV AVTFTGGGNKLT 1129 (SEQ ID NO: 7) (SEQ ID NO: 67) (SEQ ID NO: 135) DSVNN IPSGT AVNNYGQNFV 736 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 136) TISGNEY GLKNN IVNWGSNSGYALN 338 (SEQ ID NO: 8) (SEQ ID NO: 68) (SEQ ID NO: 137) DSVNN IPSGT AEDNYGQNFV 228 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 138) DSVNN IPSGT AVVNYGQNFV 218 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 139) DSVNN IPSGT AVDIYGQNFV 214 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 140) SSVPPY YTSAATLV AVSEMNYGGSQGNLI 213 (SEQ ID NO: 9) (SEQ ID NO: 69) (SEQ ID NO: 141) DSVNN IPSGT AVEGYKLS 208 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 142) VSPFSN MTFSENT VAMNRDDKII 158 (SEQ ID NO: 10) (SEQ ID NO: 70) (SEQ ID NO: 143) DSVNN IPSGT AVENYGQNFV 120 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 144) TSINN IRSNERE ATVSTSGTYKYI 109 (SEQ ID NO: 11) (SEQ ID NO: 71) (SEQ ID NO: 145) SSVSVY YLSGSTLV AVSDTGFQKLV 102 (SEQ ID NO: 7) (SEQ ID NO: 67) (SEQ ID NO: 146) DSVNN IPSGT AVDYYGQNFV 95 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 147) DSVNN IPSGT AVYNYGQNFV 71 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 148) DSVNN IPSGT AGDNYGQNFV 69 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 149) DSVNN IPSGT AVDTYGQNFV 52 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 150) DSVNN IPSGT AVANYGQNFV 50 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 151) SSNFYA MTLNGDE AFMSGYSTLT 41 (SEQ ID NO: 12) (SEQ ID NO: 72) (SEQ ID NO: 152) DRGSQS IYSNGD AVNLGGGGADGLT 41 (SEQ ID NO: 13) (SEQ ID NO: 73) (SEQ ID NO: 153) DSVNN IPSGT AVEGYSGAGSYQLT 39 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 154) DSVNN IPSGT AVDHYGQNFV 35 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 155) DSVNN IPSGT AVEPHNARLM 33 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 156) DSVNN IPSGT AVDKYGQNFV 27 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 157) TSESDYY QEAYKQQN AYRSAVTGNQFY 23 (SEQ ID NO: 14) (SEQ ID NO: 74) (SEQ ID NO: 158) DSVNN IPSGT AVHNYGQNFV 21 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 159) TSGFNG NVLDGL AVDLNSGYSTLT 17 (SEQ ID NO: 15) (SEQ ID NO: 75) (SEQ ID NO: 160) SSVSVY YLSGSTLV AVSDPGDEKLT 15 (SEQ ID NO: 7) (SEQ ID NO: 67) (SEQ ID NO: 161) TSINN IRSNERE ATVQNTGTASKLT 13 (SEQ ID NO: 11) (SEQ ID NO: 71) (SEQ ID NO: 162) NYSPAY IRENEKE ALGTEMTRS 11 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 163) TRDTTYY RNSFDEQN ALSDSEGAQKLV 10 (SEQ ID NO: 17) (SEQ ID NO: 77) (SEQ ID NO: 164) DRGSQS IYSNGD AVDGQKLL 10 (SEQ ID NO: 13) (SEQ ID NO: 73) (SEQ ID NO: 165) TSDPSYG QGSYDQQN AMREGGDDKII 9 (SEQ ID NO: 18) (SEQ ID NO: 82) (SEQ ID NO: 166) TISGNEY GLKNN IVRVASGGGADGLT 8 (SEQ ID NO: 8) (SEQ ID NO: 68) (SEQ ID NO: 167) NYSPAY IRENEKE ALRRLQNY 8 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 168) DSVNN IPSGT AVLPQGGSEKLV 8 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 169) DSVNN IPSGT AVDNRGQNFV 8 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 170) TSGFYG DALDGL ALYNFNKFY 7 (SEQ ID NO: 19) (SEQ ID NO: 83) (SEQ ID NO: 171) SIFNT LYKAGEL AGQLTLATQAN* 7 (SEQ ID NO: 20) (SEQ ID NO: 84) (SEQ ID NO: 172) DSVNN IPSGT AVDSCGQNFV 7 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 173) DSVNN IPSGT AGITMVRIL 7 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 174) YSGSPE HISR AQGSLALATQAN* 6 (SEQ ID NO: 21) (SEQ ID NO: 95) (SEQ ID NO: 175) TSESDYY QEAYKQQN ACFNSNSGYALN 6 (SEQ ID NO: 14) (SEQ ID NO: 74) (SEQ ID NO: 176) TSDPSYG QGSYDQQN AMRASGGYQKVT 6 (SEQ ID NO: 18) (SEQ ID NO: 82) (SEQ ID NO: 177) SSYSPS YTSAATLV VVSRIMEEAKEIS 6 (SEQ ID NO: 22) (SEQ ID NO: 69) (SEQ ID NO: 178) SSVPPY YTTGATLV AVSGYNNDMR 6 (SEQ ID NO: 9) (SEQ ID NO: 96) (SEQ ID NO: 179) NSAFQY TYSSGN AVGTGANNLF 6 (SEQ ID NO: 23) (SEQ ID NO: 97) (SEQ ID NO: 180) DSVNN IPSGT AG*LWSEFC 6 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 181) DSASNY IRSNVGE AASIMTC 6 (SEQ ID NO: 24) (SEQ ID NO: 80) (SEQ ID NO: 182) TSDPSYG QGSYDQQN AMDVYNQGGKLI 5 (SEQ ID NO: 18) (SEQ ID NO: 82) (SEQ ID NO: 183) SSYSPS YTSAATLV VVSGVGQNFV 5 (SEQ ID NO: 22) (SEQ ID NO: 69) (SEQ ID NO: 184) DSVNN IPSGT AVDNCGQNFV 5 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 185) DSVNN IPSGT AVDDHGQNFV 5 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 186) DSVNN IPSGT AMDNYGQNFV 5 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 187) SSVSVY YLSGSTLV AVTFAGGGNKLT 4 (SEQ ID NO: 7) (SEQ ID NO: 67) (SEQ ID NO: 188) SSVSVY YLSGSTLV AVAFTGGGNKLT 4 (SEQ ID NO: 7) (SEQ ID NO: 67) (SEQ ID NO: 189) SSVPPY YTSAATLV AVSLNDYKLS 4 (SEQ ID NO: 9) (SEQ ID NO: 69) (SEQ ID NO: 190) NSASDY IRSNMDK AEISYSSASKII 4 (SEQ ID NO: 25) (SEQ ID NO: 98) (SEQ ID NO: 191) DSVNN IPSGT TVDNYGQNFV 4 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 192) DSVNN IPSGT AVDNYSQNFV 4 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 193) DSVNN IPSGT AVDNHGQNFV 4 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 194) TSESDYY QEAYKQQN AYRSHDMR 3 (SEQ ID NO: 14) (SEQ ID NO: 74) (SEQ ID NO: 195) TSESDYY QEAYKQQN AYGGGSEKLV 3 (SEQ ID NO: 14) (SEQ ID NO: 74) (SEQ ID NO: 196) SSVSVY YLSGSTLV AVTSTGGGNKLT 3 (SEQ ID NO: 7) (SEQ ID NO: 67) (SEQ ID NO: 197) SSVSVY YLSGSTLV AVTLTGGGNKLT 3 (SEQ ID NO: 7) (SEQ ID NO: 67) (SEQ ID NO: 198) SSVSVY YLSGSTLV AVSEMNYGGSQGNLI 3 (SEQ ID NO: 7) (SEQ ID NO: 67) (SEQ ID NO: 141) NYSPAY IRENEKE APPSGSARQLT 3 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 199) DSVNN IPSGT AVNSYGQNFV 3 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 200) DSVNN IPSGT AVNDYGQNFV 3 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 201) DSVNN IPSGT AAVNYGQNFV 3 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 202) DSVNN IPSGT AANNYGQNFV 3 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 203) YGGTVN YFSGDPLV AVNRNTGNQFY 2 (SEQ ID NO: 26) (SEQ ID NO: 99) (SEQ ID NO: 204) VSPFSN MTFSENT VVSAKEAKEIS 2 (SEQ ID NO: 10) (SEQ ID NO: 70) (SEQ ID NO: 205) VSPFSN MTFSENT VVSAEGRQRLNPGEAI 2 (SEQ ID NO: 10) (SEQ ID NO: 70) (SEQ ID NO: 206) TSINN IRSNERE ATGSNDYKLS 2 (SEQ ID NO: 11) (SEQ ID NO: 71) (SEQ ID NO: 207) TSINN IRSNERE ATDGRGSYIPT 2 (SEQ ID NO: 11) (SEQ ID NO: 71) (SEQ ID NO: 208) TSINN IRSNERE ATDEDSSYKLI 2 (SEQ ID NO: 11) (SEQ ID NO: 71) (SEQ ID NO: 209) TSGFNG NVLDGL AVSDSNYQLI 2

(SEQ ID NO: 15) (SEQ ID NO: 75) (SEQ ID NO: 210) TSESDYY QEAYKQQN AYRSAGGATNKLI 2 (SEQ ID NO: 14) (SEQ ID NO: 74) (SEQ ID NO: 211) TSENNYY QEAYKQQN AFMKHSGVNDMR 2 (SEQ ID NO: 31) (SEQ ID NO: 74) (SEQ ID NO: 212) TISGNEY GLKNN IVSWGSNSGYALN 2 (SEQ ID NO: 8) (SEQ ID NO: 68) (SEQ ID NO: 213) TISGNEY GLKNN ICSGNTPLV 2 (SEQ ID NO: 8) (SEQ ID NO: 68) (SEQ ID NO: 214) TISGNEY GLKNN IANWGSNSGYALN 2 (SEQ ID NO: 8) (SEQ ID NO: 68) (SEQ ID NO: 215) SSVSVY YLSGSTLV AVTYTGGGNKLT 2 (SEQ ID NO: 7) (SEQ ID NO: 67) (SEQ ID NO: 216) SSVSVY YLSGSTLV AVTFMGGGNKLT 2 (SEQ ID NO: 7) (SEQ ID NO: 67) (SEQ ID NO: 217) SSVSVY YLSGSTLV AVTFKGGGNKLT 2 (SEQ ID NO: 7) (SEQ ID NO: 67) (SEQ ID NO: 218) SSVSVY YLSGSTLV AVSDRGETSW 2 (SEQ ID NO: 7) (SEQ ID NO: 67) (SEQ ID NO: 219) SSVSVY YLSGSTLV AVSDAGFQKLV 2 (SEQ ID NO: 7) (SEQ ID NO: 67) (SEQ ID NO: 220) SSVSVY YLSGSTLV AATFTGGGNKLT 2 (SEQ ID NO: 7) (SEQ ID NO: 67) (SEQ ID NO: 221) SSVPPY YTSAATLV AVSGMNYGGSQGNLI 2 (SEQ ID NO: 9) (SEQ ID NO: 69) (SEQ ID NO: 222) NYSPAY IRENEKE APYTGRRALT 2 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 223) DSVNN IPSGT VVDNYGQNFV 2 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 224) DSVNN IPSGT VADNYGQNFV 2 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 225) DSVNN IPSGT TADNYGQNFV 2 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 226) DSVNN IPSGT CG*LWSEFC 2 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 227) DSVNN IPSGT AWITMVRIL 2 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 228) DSVNN IPSGT AVSNYGQNFV 2 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 229) DSVNN IPSGT AVSNDYKLS 2 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 230) DSVNN IPSGT AVDS*GQNFV 2 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 231) DSVNN IPSGT AVDNYVRIL 2 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 232) DSVNN IPSGT AVDNHSQNFV 2 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 233) DRVSQS IYSNGD AVFGSNTGKLI 2 (SEQ ID NO: 32) (SEQ ID NO: 73) (SEQ ID NO: 234) ATGYPS ATKADDK ALRSNDYKLS 2 (SEQ ID NO: 33) (SEQ ID NO: 100) (SEQ ID NO: 235) AVRIAFWGLPESY 2 (SEQ ID NO: 236) TCRbeta SGHNT YYREEE ASSLAGYEQY 570775 Donor Y (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 237) (CS9.3) SGHDT YYEEEE ASSLGQGKH*SF 21232 (SEQ ID NO: 35) (SEQ ID NO: 102) (SEQ ID NO: 238) SQVTM ANQGSEA SVEGGSSGANVLT 15824 (SEQ ID NO: 36) (SEQ ID NO: 90) (SEQ ID NO: 239) SGHNT YYREEE ASSSAGYEQY 1390 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 240) SGHNT YYREEE ASSLAGCEQY 1191 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 241) SGHDY FNNNVP ASTSWGVSYNEQF 1176 (SEQ ID NO: 37) (SEQ ID NO: 103) (SEQ ID NO: 242) SGHNT YYREEE ASSLASYEQY 1036 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 243) SGHNT YYREEE ASSLAGHEQY 1003 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 244) KGHSH LQKENI ASSPPEGFGNEQF 612 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 245) SGHNT YYREEE ASSLTGYEQY 546 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 246) DFQATT SNEGSKA SANLAKSSYNEQF 265 (SEQ ID NO: 39) (SEQ ID NO: 89) (SEQ ID NO: 247) DFQATT SNEGSKA SAPRDPDADTQY 231 (SEQ ID NO: 39) (SEQ ID NO: 89) (SEQ ID NO: 248) GTSNPN SVGIG AWDRTGEVEQY 186 (SEQ ID NO: 40) (SEQ ID NO: 104) (SEQ ID NO: 249) SGHNT YYREEE ASS*AGYEQY 174 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 250) SGHNT YYREEE AAAWPATSS 151 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 251) SGHNT YYREEE ASSMAGYEQY 140 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 252) GTSNPN SVGIG AWSFHPGLAAYNEQF 108 (SEQ ID NO: 40) (SEQ ID NO: 104) (SEQ ID NO: 253) SGHDN FVKESK ASSQLRGGSPLH 100 (SEQ ID NO: 41) (SEQ ID NO: 105) (SEQ ID NO: 254) MNHEY SVGAGI ASSGQGGSNTEAF 98 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 255) SGHDT YYEEEE ASSLGQGRH*SF 89 (SEQ ID NO: 35) (SEQ ID NO: 102) (SEQ ID NO: 256) SGHVS FNYEAQ ASSLAEDTQY 81 (SEQ ID NO: 43) (SEQ ID NO: 106) (SEQ ID NO: 257) SGHNT YYREEE ASSWAGYEQY 70 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 258) SGHDT YYEEEE ASSLGRGKH*SF 70 (SEQ ID NO: 35) (SEQ ID NO: 102) (SEQ ID NO: 259) SGHNT YYREEE ASSLAGFEQY 68 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 260) SGHNT YYREEE ASSLAG*EQY 59 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 261) SGHDT YYEEEE ASSLGQGKR*SF 58 (SEQ ID NO: 35) (SEQ ID NO: 102) (SEQ ID NO: 262) SGHDT YYEEEE ASSLGQGEH*SF 57 (SEQ ID NO: 35) (SEQ ID NO: 102) (SEQ ID NO: 263) GTSNPN SVGIG AYSTGYFGYT 54 (SEQ ID NO: 40) (SEQ ID NO: 104) (SEQ ID NO: 264) SQVTM ANQGSEA SVGGGSSGANVLT 49 (SEQ ID NO: 36) (SEQ ID NO: 90) (SEQ ID NO: 265) SGHNT YYREEE ASSVAGYEQY 49 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 266) SQVTM ANQGSEA SVEGGSPGANVLT 47 (SEQ ID NO: 36) (SEQ ID NO: 90) (SEQ ID NO: 267) SGHDT YYEEEE ASSSGQGKH*SF 47 (SEQ ID NO: 35) (SEQ ID NO: 102) (SEQ ID NO: 268) SNHLY FYNNEI ASSESRYGRDTDTQY 39 (SEQ ID NO: 44) (SEQ ID NO: 94) (SEQ ID NO: 269) MNHEY SVGEGT ASSYSYSTGPELNTEAF 38 (SEQ ID NO: 42) (SEQ ID NO: 91) (SEQ ID NO: 270) SQVTM ANQGSEA SVEGGPSGANVLT 36 (SEQ ID NO: 36) (SEQ ID NO: 90) (SEQ ID NO: 271) SGHNT YYREEE ASSLAGNEQY 34 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 272) SGHNT YYREEE ASSLAAYEQY 33 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 273) SGHNT YYREEE ASSLSFDSEQY 32 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 274) SGHDT YYEEEE ASSLSQGKH*SF 32 (SEQ ID NO: 35) (SEQ ID NO: 102) (SEQ ID NO: 275) SGHNT YYREEE ASSFAGYEQY 31 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 276) SQVTM ANQGSEA SARQGLTEAF 27 (SEQ ID NO: 36) (SEQ ID NO: 90) (SEQ ID NO: 277) GTSNPN SVGIG AWSVLYGTEY 27 (SEQ ID NO: 40) (SEQ ID NO: 104) (SEQ ID NO: 278) SGHNT YYREEE ASSLAGSEQY 26 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 279) SQVTM ANQGSEA SVEGDPLGPTS* 24 (SEQ ID NO: 36) (SEQ ID NO: 90) (SEQ ID NO: 280) SGHNT YYREEE ASSLSGYEQY 24 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 281) SQVTM ANQGSEA SVEEGSSGANVLT 22 (SEQ ID NO: 36) (SEQ ID NO: 90) (SEQ ID NO: 282) SGHNT YYREEE ASSLPGYEQY 21 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 283) LNHNV YYDKDF ATSREGTGENIQY 21 (SEQ ID NO: 45) (SEQ ID NO: 107) (SEQ ID NO: 284) SGHDT YYEEEE ASSFSIRASYEQY 19 (SEQ ID NO: 35) (SEQ ID NO: 102) (SEQ ID NO: 285) SGHNT YYREEE ASSLAGDEQY 17 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 286) ASSVASTGELF 16 (SEQ ID NO: 287) SGHDT YYEEEE ASSLGRGNTEAF 15 (SEQ ID NO: 35) (SEQ ID NO: 102) (SEQ ID NO: 288) SGHAT FQNNGV ASSPIRREGEQY 15 (SEQ ID NO: 46) (SEQ ID NO: 108) (SEQ ID NO: 289) KGHSH LQKENI ASFVYSAGDSYNEQF 15 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 290) DFQATT SNEGSKA SARNRVYEQY 15 (SEQ ID NO: 39) (SEQ ID NO: 89) (SEQ ID NO: 291) SGHDT YYEEEE ASSLGQGNTEAF 14 (SEQ ID NO: 35) (SEQ ID NO: 102) (SEQ ID NO: 292) MNHEY SVGAGI ASSPPGENEQY 12 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 293)

LNHDA SQIVND ASTDTDLGEQY 12 (SEQ ID NO: 47) (SEQ ID NO: 109) (SEQ ID NO: 294) SQVTM ANQGSEA SVERGSSGANVLT 11 (SEQ ID NO: 36) (SEQ ID NO: 90) (SEQ ID NO: 295) SGHNT YYREEE ASSLGQGKH*SF 11 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 238) SGHDT YYEEEE ASSLGQGNH*SF 11 (SEQ ID NO: 35) (SEQ ID NO: 102) (SEQ ID NO: 296) SGHDT YYEEEE ASSLARGNTEAF 11 (SEQ ID NO: 35) (SEQ ID NO: 102) (SEQ ID NO: 297) SGHDT YYEEEE AAAWARGNTEAF 11 (SEQ ID NO: 35) (SEQ ID NO: 102) (SEQ ID NO: 298) ENHRY SYGVKD ALSDSGTIYEQY 11 (SEQ ID NO: 48) (SEQ ID NO: 110) (SEQ ID NO: 299) WSHSY SAAADI ASSVPLEGGSGPQDTQY 10 (SEQ ID NO: 49) (SEQ ID NO: 111) (SEQ ID NO: 300) SGHDT YYEEEE ASSLGQGKY*SF 10 (SEQ ID NO: 35) (SEQ ID NO: 102) (SEQ ID NO: 301) LGHNT FRNRAP ASGLYNRGNEQF 10 (SEQ ID NO: 50) (SEQ ID NO: 112) (SEQ ID NO: 302) SQVTM ANQGSEA SVEGGSSGPTS* 9 (SEQ ID NO: 36) (SEQ ID NO: 90) (SEQ ID NO: 303) SGHNT YYREEE ASGLAGYEQY 9 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 304) KGHSH LQKENI ASSRTRYTDTQY 9 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 305) SGHNT YYREEE ASNLAGYEQY 8 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 306) SGHDY FNNNVP ASASWGVSYNEQF 8 (SEQ ID NO: 37) (SEQ ID NO: 103) (SEQ ID NO: 307) SGHDT YYEEEE ASSLGQGETLKL 8 (SEQ ID NO: 35) (SEQ ID NO: 102) (SEQ ID NO: 308) SQVTM ANQGSEA SVVGGSSGANVLT 7 (SEQ ID NO: 36) (SEQ ID NO: 90) (SEQ ID NO: 309) SQVTM ANQGSEA SVGANVAGGKETQY 7 (SEQ ID NO: 36) (SEQ ID NO: 90) (SEQ ID NO: 310) ASSVTGTVNTEAF 7 (SEQ ID NO: 311) SQVTM ANQGSEA SVKGGSSGANVLT 6 (SEQ ID NO: 36) (SEQ ID NO: 90) (SEQ ID NO: 312) SQVTM ANQGSEA SVEGGSTGANVLT 6 (SEQ ID NO: 36) (SEQ ID NO: 90) (SEQ ID NO: 313) SGHDT YYGEEE ASSLGQGIH*SF 6 (SEQ ID NO: 35) (SEQ ID NO: 113) (SEQ ID NO: 314) SGHDT YYEEEE ASSLGQGKL*SF 6 (SEQ ID NO: 35) (SEQ ID NO: 102) (SEQ ID NO: 315) SGHDN FVKESK ASSQDIEV*EAF 6 (SEQ ID NO: 41) (SEQ ID NO: 105) (SEQ ID NO: 316) LGHDT YNNKEL ASSLRLNTEAF 6 (SEQ ID NO: 51) (SEQ ID NO: 114) (SEQ ID NO: 317) ASSVEAGVSGNTIY 6 (SEQ ID NO: 318) SQVTM ANQGSEA SVVRQGHYEAF 5 (SEQ ID NO: 36) (SEQ ID NO: 90) (SEQ ID NO: 319) SQVTM ANQGSEA SVEGGSFGANVLT 5 (SEQ ID NO: 36) (SEQ ID NO: 90) (SEQ ID NO: 320) SNHLY FYNNEI ASSPGRILTDTQY 5 (SEQ ID NO: 44) (SEQ ID NO: 94) (SEQ ID NO: 321) SGHNT YYREEE ASSLADYEQY 5 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 322) MNHEY SVGAGI ASSGGLNQPQH 5 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 323) MDHEN SYDVKM ASKVQGSEDTQY 5 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 324) KGHSH LQKENI ASSPPGGFGNEQF 5 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 325) SNHLY FYNNEI ASSGAGQGSSYEQY 4 (SEQ ID NO: 44) (SEQ ID NO: 94) (SEQ ID NO: 326) SGHNT YYREEE ASSLGQGEH*SF 4 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 263) SGHNT YYREEE ASSLASCEQY 4 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 327) SGHNT YYREEE ASSLAGYRQY 4 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 328) SGHNT YYREEE ASSLAGYKQY 4 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 329) SGHDY FNNNVP ASTSWASPTMSS 4 (SEQ ID NO: 37) (SEQ ID NO: 103) (SEQ ID NO: 330) SGHDY FNNNVP ASTSRGVSYNEQF 4 (SEQ ID NO: 37) (SEQ ID NO: 103) (SEQ ID NO: 331) SGHDT YYEEEE ASSFGQGKH*SF 4 (SEQ ID NO: 35) (SEQ ID NO: 102) (SEQ ID NO: 332) SGHDT YYEEEE ASS*GQGKH*SF 4 (SEQ ID NO: 35) (SEQ ID NO: 102) (SEQ ID NO: 333) SGHNT YYREEE ASSLVGHEQY 3 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 334) SGHNT YYREEE ASSLGRGKH*SF 3 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 259) SGHNT YYREEE ASSLAGHGQY 3 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 335) SGHNT YYREEE ASNSAGYEQY 3 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 336) SGHNT YYREEE ASGSAGYEQY 3 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 337) SGHDY FNNNVP ASTSWGISYNEQF 3 (SEQ ID NO: 37) (SEQ ID NO: 103) (SEQ ID NO: 338) SGHDY FNNNVP ASTSWGASYNEQF 3 (SEQ ID NO: 37) (SEQ ID NO: 103) (SEQ ID NO: 339) SGHDY FNNNVP ASTS*GVSYNEQF 3 (SEQ ID NO: 37) (SEQ ID NO: 103) (SEQ ID NO: 340) SGHDY FNNNVP AGTSWGVSYNEQF 3 (SEQ ID NO: 37) (SEQ ID NO: 103) (SEQ ID NO: 341) SGHDT YYEEEE ASSVGQGKH*SF 3 (SEQ ID NO: 35) (SEQ ID NO: 102) (SEQ ID NO: 342) SGHDT YYEEEE ASSMGQGKH*SF 3 (SEQ ID NO: 35) (SEQ ID NO: 102) (SEQ ID NO: 343) SGHDT YYEEEE ASSLCQGKH*SF 3 (SEQ ID NO: 35) (SEQ ID NO: 102) (SEQ ID NO: 344) MNHNS SASEGT ASRGLAGFNEQF 3 (SEQ ID NO: 53) (SEQ ID NO: 92) (SEQ ID NO: 345) MNHEY SMNVEV ASSLMRVGFRTDTQY 3 (SEQ ID NO: 42) (SEQ ID NO: 115) (SEQ ID NO: 346) MGHRA YSYEKL ASSQDELAGRTQY 3 (SEQ ID NO: 54) (SEQ ID NO: 116) (SEQ ID NO: 347) MDHEN SYDVKM ASTNSLTSTDTQY 3 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 348) KGHSH LQKENI ASSPPEGLGNEQF 3 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 349) ENHRY SYGVKD AISRADQETQY 3 (SEQ ID NO: 48) (SEQ ID NO: 110) (SEQ ID NO: 350) DFQATT SNEGSKA SARDRGATGELF 3 (SEQ ID NO: 39) (SEQ ID NO: 89) (SEQ ID NO: 351) SQVTM ANQGSEA SVEGGSAGANVLT 2 (SEQ ID NO: 36) (SEQ ID NO: 90) (SEQ ID NO: 352) SQVTM ANQGSEA SARQGRTEAF 2 (SEQ ID NO: 36) (SEQ ID NO: 90) (SEQ ID NO: 353) SGHYY FNNNVP ASTSWGVPYNEQF 2 (SEQ ID NO: 55) (SEQ ID NO: 103) (SEQ ID NO: 354) SGHNT YYREEE ASSLTSYEQY 2 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 355) SGHNT YYREEE ASSLTGCEQY 2 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 356) SGHNT YYREEE ASSLGQRKH*SF 2 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 357) SGHNT YYREEE ASSLGQGRH*SF 2 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 256) SGHNT YYREEE ASSLGQGKR*SF 2 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 262) SGHNT YYREEE ASSLAGYK*Y 2 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 358) SGHNT YYREEE ASGMAGYEQY 2 (SEQ ID NO: 34) (SEQ ID NO: 101) (SEQ ID NO: 359) SGHDT YYEEEE ASSWARGNTEAF 2 (SEQ ID NO: 35) (SEQ ID NO: 102) (SEQ ID NO: 360) SGHDT YYEEEE ASSLGQETLKL 2 (SEQ ID NO: 35) (SEQ ID NO: 102) (SEQ ID NO: 361) SEHNR FQNEAQ ASTLYEKLF 2 (SEQ ID NO: 56) (SEQ ID NO: 117) (SEQ ID NO: 362) PGHNT YYREEE ASDLAGYEQY 2 (SEQ ID NO: 57) (SEQ ID NO: 101) (SEQ ID NO: 363) MDHEN SYDVKM ASVGTGNVDEQY 2 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 364) KGHSH LQKENI ASSPPEGSGNEQF 2 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 365) KGHSH LQKENI ASSPPEGFSNEQF 2 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 366) DFQATT SNEGSKA SANLARSSYNEQF 2 (SEQ ID NO: 39) (SEQ ID NO: 89) (SEQ ID NO: 367) DFQATT SNEGSKA SALDLAGSQETQY 2 (SEQ ID NO: 39) (SEQ ID NO: 89) (SEQ ID NO: 368) TCRalpha DSVNN IPSGT AVELFAAGNKLT 66076 Donor R (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 369) (CS9.3) SVFSS VVTGGEV AGAVTGQLQQIL 191 (SEQ ID NO: 58) (SEQ ID NO: 118) (SEQ ID NO: 370) DSVNN IPSGT AVGLFAAGNKLT 184 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 371)

DSVNN IPSGT AVELLAAGNKLT 161 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 372) DSVNN IPSGT AVELSAAGNKLT 126 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 373) DSVNN IPSGT AVKLFAAGNKLT 72 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 374) DSVNN IPSGT AVELFTAGNKLT 65 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 375) DSVNN IPSGT AVELFATGNKLT 58 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 376) DSVNN IPSGT AVELFVAGNKLT 42 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 377) DSVNN IPSGT AVELFAAGNKL (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 378) DSVNN IPSGT AVVLFAAGNKLT (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 379) DSVNN IPSGT AVSYLLQATS* (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 380) DSVNN IPSGT AV*LFAAGNKLT (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 381) DSVNN IPSGT AVELFASGNKLT (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 382) DSVNN IPSGT AVELFAAATS* (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 383) DSVNN IPSGT AVDLFAAGNKLT (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 384) DSVNN IPSGT AVELFDAGNKLT (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 385) IFSNGE AASEGNYNVLY (SEQ ID NO: 85) (SEQ ID NO: 386) DSVNN IPSGT AVEVFAAGNKLT (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 387) DSVNN IPSGT AVELFSAGNKLT (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 388) DSVNN IPSGT AVALFAAGNKLT (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 389) DSVNN IPSGT AVEIFAAGNKLT (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 390) DSVNN IPSGT AVELFPAGNKLT (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 391) DSVNN IPSGT AVELFGAGNKLT (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 392) DSVNN IPSGT AVELFAEATS* (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 393) TCRbeta MDHEN SYDVKM ASSLISGSSYEQY 111183 Donor R (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 394) (CS9.3) LNHDA SQIVND ASSIEGQK*TLKL 7924 (SEQ ID NO: 47) (SEQ ID NO: 109) (SEQ ID NO: 395) MDHEN SYDVKM ASSLISGSPYEQY 368 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 396) MDHEN SYDVKM ASGLISGSSYEQY 312 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 397) MDHEN SYDVKM ASSLIGGSSYEQY 293 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 398) MDHEN SYDVKM ASSLVSGSSYEQY 233 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 399) MDHEN SYDVKM ASSPISGSSYEQY 230 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 400) MDHEN SYDVKM ASSLISGGSYEQY 222 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 401) MDHEN SYDVKM ASNLISGSSYEQY 98 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 402) MDHEN SYDVKM ASSLISGSFYEQY 65 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 403) MDHEN SYDVKM ASSLISGSTYEQY 45 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 404) LNHDA SQIVND ASSIEGQK*ALKL 43 (SEQ ID NO: 47) (SEQ ID NO: 109) (SEQ ID NO: 405) MDHEN SYDVKM ASRLISGSSYEQY 38 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 406) LNHDA SQIVND ASSIEGQKWTLKL 33 (SEQ ID NO: 47) (SEQ ID NO: 109) (SEQ ID NO: 407) MDHEN SYDVKM AAV**VVAPTSS 30 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 408) LNHDA SQIVND ASSIGGQK*TLKL 27 (SEQ ID NO: 47) (SEQ ID NO: 109) (SEQ ID NO: 409) LNHDA SQIVND ASSIEGQR*TLKL 26 (SEQ ID NO: 47) (SEQ ID NO: 109) (SEQ ID NO: 410) LNHDA SQIVND ASSIEGRK*TLKL 23 (SEQ ID NO: 47) (SEQ ID NO: 109) (SEQ ID NO: 411) LNHDA SQIVND ASSTEGQK*TLKL 17 (SEQ ID NO: 47) (SEQ ID NO: 109) (SEQ ID NO: 412) MDHEN SYDVKM ASSLISVAPTSS 16 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 413) LNHDA SQIVND ASSIEGQE*TLKL 16 (SEQ ID NO: 47) (SEQ ID NO: 109) (SEQ ID NO: 414) MDHEN SYDVKM ARSLISGSSYEQY 15 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 415) LNHDA SQIVND ASSMEGQK*TLKL 15 (SEQ ID NO: 47) (SEQ ID NO: 109) (SEQ ID NO: 416) LNHDA SQIVND ASSIEEQK*TLKL 15 (SEQ ID NO: 47) (SEQ ID NO: 109) (SEQ ID NO: 417) LNHDA SQIVND ASSIEGQKRTLKL 14 (SEQ ID NO: 47) (SEQ ID NO: 109) (SEQ ID NO: 418) MDHEN SYDVKM ASSLISGSAYEQY 13 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 419) MDHEN SYDVKM ASCLISGSSYEQY 9 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 420) MDHEN SYDVKM ASSLISGRSYEQY 8 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 421) MDHEN SYDVKM ASSLISGSYYEQY 6 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 422) LNHDA SQIVND ASSIEGQKCTLKL 6 (SEQ ID NO: 47) (SEQ ID NO: 109) (SEQ ID NO: 423) MDHEN SYDVKM ASSLSGSSYEQY 4 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 424) MDHEN SYDVKM ASSLISGSSTSS 4 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 425) MDHEN SYDVKM ASSLISGSSREQY 4 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 426) MDHEN SYDVKM ASILISGSSYEQY 3 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 427) LNHDA SQIVND ASSKEGQK*TLKL 3 (SEQ ID NO: 47) (SEQ ID NO: 109) (SEQ ID NO: 428) MDHEN SYDVKM ASTLISGSSYEQY 2 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 429) MDHEN SYDVKM ASSPISGSPYEQY 2 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 430) MDHEN SYDVKM ASSLVSGNSYEQY 2 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 431) MDHEN SYDVKM ASSLISGSCYEQY 2 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 432) MDHEN SYDVKM ASGPISGSSYEQY 2 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 433) MDHEN SYDVKM ASGLISGGSYEQY 2 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 434) MDHEN SYDVKM ASGLIGGSSYEQY 2 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 435) LNHDA SQVVND ASSIEGQK*H*SF 2 (SEQ ID NO: 47) (SEQ ID NO: 119) (SEQ ID NO: 436) LNHDA SQIVND ASSREGQK*TLKL 2 (SEQ ID NO: 47) (SEQ ID NO: 109) (SEQ ID NO: 437) LNHDA SQIVND ASSIKGQK*TLKL 2 (SEQ ID NO: 47) (SEQ ID NO: 109) (SEQ ID NO: 438) LNHDA SQIVND ASSIEGQKGTLKL 2 (SEQ ID NO: 47) (SEQ ID NO: 109) (SEQ ID NO: 439) TCRalpha NYSPAY IRENEKE APPSGSARQLT 687383 Donor Y (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 199) (CS11.6) NYSPAY IRENEKE APPPGSARQLT 2142 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 440) SSVSVY YLSGSTLV AVMNAGKST 1155 (SEQ ID NO: 7) (SEQ ID NO: 67) (SEQ ID NO: 441) NYSPAY IRENEKE ALPSGSARQLT 739 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 442) NYSPAY IRENEKE APPSSSARQLT 652 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 443) NYSPAY IRENEKE TPPSGSARQLT 644 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 444) NYSPAY IRENEKE VPPSGSARQLT 450 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 445) NYSPAY IRENEKE APSSGSARQLT 439 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 446) NYSPAY IRENEKE APPFGSARQLT 411 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 447) NYSPAY IRENEKE APPTGSARQLT 258 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 448) NSASDY IRSNMDK AENEDYGQNFV 142 (SEQ ID NO: 25) (SEQ ID NO: 98) (SEQ ID NO: 449) NYSPAY IRENEKE APPSGSARQL 123 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 450) NYSPAY IRENEKE APPSGSARQLTFGSGTQL 81 (SEQ ID NO: 16) (SEQ ID NO: 76) TVLPEHIKKRGEVTKGSL L*GIKHCDTHGRRKQTH (SEQ ID NO: 451) NYSPAY IRENEKE AQPSGSARQLT 77 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 452) NYSPAY IRENEKE APPSCSARQLT 57

(SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 453) NYSPAY IRENEKE APPAGSARQLT 56 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 454) NYSPAY IRENEKE APPYGSARQLT 34 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 455) NYSPAY IRENEKE DPPSGSARQLT 30 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 456) NYSPAY IRENEKE GPPSGSARQLT 29 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 457) DRGSQS IYSNGD AVNIGGSQGNLI 19 (SEQ ID NO: 13) (SEQ ID NO: 73) (SEQ ID NO: 458) NYSPAY IRENEKE SPPSGSARQLT 18 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 459) NYSPAY IRENEKE APPSRSARQLT 16 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 460) NYSPAY IRENEKE ARPLVLQGN* 14 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 461) NYSPAY IRENEKE PPPSGSARQLT 13 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 462) NYSPAY IRENEKE APPSDSARQLT 7 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 463) DRGSQS IYSNGD AVYSGYSTLT 7 (SEQ ID NO: 13) (SEQ ID NO: 73) (SEQ ID NO: 464) YGGTVN YFSGDPLV AVNARDSGTYKYI 6 (SEQ ID NO: 26) (SEQ ID NO: 99) (SEQ ID NO: 465) NTAFDY IRPDVS AAPGECWQQP*AD 6 (SEQ ID NO: 27) (SEQ ID NO: 120) (SEQ ID NO: 466) TSINN IRSNERE ARTGYSGGGADGLT 4 (SEQ ID NO: 11) (SEQ ID NO: 71) (SEQ ID NO: 467) TSESDYY QEAYKQQN AYDQGGSEKLV 4 (SEQ ID NO: 14) (SEQ ID NO: 74) (SEQ ID NO: 468) TSDQSYG QGSYDEQN AMSFRGGYQKVT 4 (SEQ ID NO: 28) (SEQ ID NO: 121) (SEQ ID NO: 469) NYSPAY IRENEKE APPSGPARQLT 4 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 470) NYSPAY IRENEKE APPSDPARQLT 4 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 471) NYSPAY IRENEKE APPCGSARQLT 4 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 472) DSAIYN IQSSQRE AVRPIEHR*PVL 4 (SEQ ID NO: 29) (SEQ ID NO: 122) (SEQ ID NO: 473) YSGSPE HISR ALRSGGYQKVT 3 (SEQ ID NO: 21) (SEQ ID NO: 95) (SEQ ID NO: 474) YGATPY YFSGDTLV AVGAGGKLI 3 (SEQ ID NO: 30) (SEQ ID NO: 123) (SEQ ID NO: 475) SSVSVY YLSGSTLV AVTNAGKST 3 (SEQ ID NO: 7) (SEQ ID NO: 67) (SEQ ID NO: 476) SSVSVY YLSGSTLV AVMSAGKST 3 (SEQ ID NO: 7) (SEQ ID NO: 67) (SEQ ID NO: 477) SSVSVY YLSGSTLV AVMDAGKST 3 (SEQ ID NO: 7) (SEQ ID NO: 67) (SEQ ID NO: 478) NYSPAY IRENEKE PLVLQGN* 3 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 479) NYSPAY IRENEKE APPSGSARQLTFGSGTQL 3 (SEQ ID NO: 16) (SEQ ID NO: 76) TVLPEHIKKRGEVTKGSL L*GIKHCETHGRRKQTH (SEQ ID NO: 480) NYSPAY IRENEKE APPPDSARQLT 3 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 481) NSAFQY TYSSGN AMSLDNYGQNFV 3 (SEQ ID NO: 23) (SEQ ID NO: 97) (SEQ ID NO: 482) TSENNYY QEAYKQQN AFILQGAQKLV 2 (SEQ ID NO: 31) (SEQ ID NO: 74) (SEQ ID NO: 483) TRDTTYY RNSFDEQN ALELSGYALN 2 (SEQ ID NO: 17) (SEQ ID NO: 77) (SEQ ID NO: 484) TISGTDY GLTSN IWLRADLKSW 2 (SEQ ID NO: 79) (SEQ ID NO: 78) (SEQ ID NO: 485) SSVSVY YLSGSTLV AYGSSNTGKLI 2 (SEQ ID NO: 7) (SEQ ID NO: 67) (SEQ ID NO: 486) SSVSVY YLSGSTLV AVSARRQNFV 2 (SEQ ID NO: 7) (SEQ ID NO: 67) (SEQ ID NO: 487) SSVSVY YLSGSTLV AVRNAGKST 2 (SEQ ID NO: 7) (SEQ ID NO: 67) (SEQ ID NO: 488) SSVSVY YLSGSTLV AMMNAGKST 2 (SEQ ID NO: 7) (SEQ ID NO: 67) (SEQ ID NO: 489) NYSPAY IRENEKE VPSSGSARQLT 2 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 490) NYSPAY IRENEKE TPPFGSARQLT 2 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 491) NYSPAY IRENEKE TPLSGSARQLT 2 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 492) NYSPAY IRENEKE SALWFCKATD 2 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 493) NYSPAY IRENEKE ATPPGSARQLT 2 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 494) NYSPAY IRENEKE ASPPGSARQLT 2 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 495) NYSPAY IRENEKE APSSAGNNRKLI 2 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 496) NYSPAY IRENEKE APPSVLQGN* 2 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 497) NYSPAY IRENEKE APPSGSARQLTFGSGTQL 2 (SEQ ID NO: 16) (SEQ ID NO: 76) AVLPEHIKKRGEVTKGSL L*GIKHCDTHGRRKQTH (SEQ ID NO: 498) NYSPAY IRENEKE APPSGSAGNW 2 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 499) NYSPAY IRENEKE APPSGLARQLT 2 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 500) NYSPAY IRENEKE APPPGSARQL 2 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 501) NYSPAY IRENEKE APPLGSARQLT 2 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 502) NYSPAY IRENEKE ALPPGSARQLT 2 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 503) NYSPAY IRENEKE ALDLTGNQFY 2 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 504) NSAFQY TYSSGN AASLSNFGNEKLT 2 (SEQ ID NO: 23) (SEQ ID NO: 97) (SEQ ID NO: 505) DSVNN IPSGT AVDNYGQNFV 2 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 130) DSASNY IRSNVGE AASFSDQTGANNLF 2 (SEQ ID NO: 24) (SEQ ID NO: 80) (SEQ ID NO: 506) TCRbeta KGHSH LQKENI ASSPPEGFGNEQF 587843 Donor Y (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 245) (CS11.6) KGHSH LQKENI ASSPPGGFGNEQF 1829 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 325) KGHSH LQKENI ASSPPEGFGDEQF 1580 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 507) KGHSH LQKENI ASSPPEGLGNEQF 1481 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 349) KGHSH LQKENI ASSPPEGFGSEQF 1274 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 508) KGHSH LQKENI ASSPPEGSGNEQF 1083 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 365) KGHSH LQKENI ASSPPEGFDNEQF 640 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 509) KGHSH LQKENI ASSLPEGFGNEQF 491 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 510) KGHSH LQKENI ASSPLEGFGNEQF 451 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 511) KGHSH LQKENI ASSPSEGFGNEQF 381 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 512) KGHSH LQKENI ASSPPEGLAMSS 357 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 513) KGHSH LQKENI ASSHLRVLAMSS 259 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 514) MNHEY SMNVEV ASSPPGLGYEQY 186 (SEQ ID NO: 42) (SEQ ID NO: 115) (SEQ ID NO: 515) SGHRS YFSETQ ASSPRGGSYEQY 173 (SEQ ID NO: 59) (SEQ ID NO: 124) (SEQ ID NO: 516) KGHSH LQKENI ASSPPEGFGKEQF 146 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 517) ENHRY SYGVKD AIRSTASTDTQY 138 (SEQ ID NO: 48) (SEQ ID NO: 110) (SEQ ID NO: 518) KGHSH LQKENI ASSPHEGFGNEQF 130 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 519) KGHSH LQKENI AAHHLRVLAMSS 111 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 520) KGHSH LQKENI ASSPPEGFGYEQF 83 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 521) KGHSH LQKENI ASSQPEGFGNEQF 81 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 522) MRHNA SNTAGT ASRGTSVQQ*AV 57 (SEQ ID NO: 60) (SEQ ID NO: 125) (SEQ ID NO: 523) SGHDY FNNNVP ASTPSGPSTDTQY 53 (SEQ ID NO: 37) (SEQ ID NO: 103) (SEQ ID NO: 524) KGHSH LQKENI ASSPPEGFGIEQF 53 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 525) MNHEY SVGAGI ASSYSGAGGPWDTQY 49 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 526) KGHSH LQKENI ASSPPEGFGTEQF 45 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 527) ASSVEGTGTSIQY 45 (SEQ ID NO: 528) KGHSH LQKENI ASSPPEGFGHEQF 37 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 529) KGHSH LQKENI ASSPTEGFGNEQF 26

(SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 530) SGHVS FNYEAQ ASSLWGTEAF 18 (SEQ ID NO: 43) (SEQ ID NO: 106) (SEQ ID NO: 531) KGHSH LQKENI ASSRPEGFGNEQF 18 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 532) KGHSH LQKENI ASSPREGFGNEQF 18 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 533) MNHEY SMNVEV ASSLDRLYTEAF 12 (SEQ ID NO: 42) (SEQ ID NO: 115) (SEQ ID NO: 534) PRHDT FYEKMQ ASSFGTGGNTQY 9 (SEQ ID NO: 61) (SEQ ID NO: 126) (SEQ ID NO: 535) KGHSH LQKENI ASSPAEGFGNEQF 9 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 536) MNHEY SVGAGI ASSLYFGQPQH 7 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 537) MNHEY SVGAGI ASRTEAREQY 6 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 538) LGHDT YNNKEL ASSQPGQYGYT 6 (SEQ ID NO: 51) (SEQ ID NO: 114) (SEQ ID NO: 539) KGHSH LQKENI ASSPPEGFGDGQF 6 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 540) SGHDY FNNNVP ASRLGHQPQH 5 (SEQ ID NO: 37) (SEQ ID NO: 103) (SEQ ID NO: 541) KGHSH LQKENI ASSPPKGFGNEQF 5 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 542) KGHSH LQKENI ASSPPEGFGNRQF 5 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 543) KGHSH LQKENI ASPPPEGFGNEQF 5 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 544) SQVTM ANQGSEA SVGVTGGTITPHEQY 4 (SEQ ID NO: 36) (SEQ ID NO: 90) (SEQ ID NO: 545) SGHRS YFSETQ ASSDRDRDG*RARGGEQF 4 (SEQ ID NO: 59) (SEQ ID NO: 124) (SEQ ID NO: 546) KGHSH LQKENI ASSPPKGLGNEQF 4 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 547) KGHSH LQKENI ASSPPESLGNEQF 4 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 548) KGHSH LQKENI ASSPPEGFGNKQF 4 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 549) GTSNPN SVGIG AWRPGYMNTEAF 4 (SEQ ID NO: 40) (SEQ ID NO: 104) (SEQ ID NO: 550) ENHRY SYGVKD AISEWASGRPSYEQY 4 (SEQ ID NO: 48) (SEQ ID NO: 110) (SEQ ID NO: 551) ASSALAGDTYEQY 4 (SEQ ID NO: 552) SGHRS YFSETQ ASSPRRGSYEQY 3 (SEQ ID NO: 59) (SEQ ID NO: 124) (SEQ ID NO: 553) SGHRS YFSETQ ASSPRGAPTSS 3 (SEQ ID NO: 59) (SEQ ID NO: 124) (SEQ ID NO: 554) MDHEY SVGAGI ASSYSPGNHQPQH 3 (SEQ ID NO: 62) (SEQ ID NO: 88) (SEQ ID NO: 555) KGHSH LQKENI ASSPSEGFDNEQF 3 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 556) KGHSH LQKENI ASSPPEGLSNEQF 3 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 557) KGHSH LQKENI ASSPPEGFSSEQF 3 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 558) KGHSH LQKENI ASSPPEGFGNE*F 3 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 559) KGHSH LQKENI ASSPPEDFGNEQF 3 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 560) ENHRY SYGVKD AIRSTASADTQY 3 (SEQ ID NO: 48) (SEQ ID NO: 110) (SEQ ID NO: 561) SNHLY FYNNEI ATTRTSGSNEQF 2 (SEQ ID NO: 44) (SEQ ID NO: 94) (SEQ ID NO: 562) SGHRS YFSETQ ASSPRGGLLRAV 2 (SEQ ID NO: 59) (SEQ ID NO: 124) (SEQ ID NO: 563) MNHEY SVGEGT ASSYGLAHSYEQY 2 (SEQ ID NO: 42) (SEQ ID NO: 91) (SEQ ID NO: 564) MNHEY SMNVEV ATLQGPNEQF 2 (SEQ ID NO: 42) (SEQ ID NO: 115) (SEQ ID NO: 565) MDHEN SYDVKM ASSSSVLRARTEAF 2 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 566) LNHDA SQIVND ASSIFLGDNTGELF 2 (SEQ ID NO: 47) (SEQ ID NO: 109) (SEQ ID NO: 567) KGHSR LQKENI ASLPPEGFGNEQF 2 (SEQ ID NO: 63) (SEQ ID NO: 81) (SEQ ID NO: 568) KGHSH LQKENI ASSPSEDFGNEQF 2 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 569) KGHSH LQKENI ASSPPKGSGNEQF 2 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 570) KGHSH LQKENI ASSPPGGFDNEQF 2 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 571) KGHSH LQKENI ASSPPEGFSNEQF 2 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 366) KGHSH LQKENI ASSPPEGFNNEQF 2 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 572) KGHSH LQKENI ASSPPEGFGYGQF 2 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 573) KGHSH LQKENI ASSPPEGFGNGQF 2 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 574) KGHSH LQKENI ASSPPEDSGNEQF 2 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 575) KGHSH LQKENI ASSPPEDFDNEQF 2 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 576) KGHSH LQKENI ASSPLKGFGNEQF 2 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 577) KGHSH LQKENI ASSPLGGFGNEQF 2 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 578) KGHSH LQKENI ASSPLEGFSNEQF 2 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 579) KGHSH LQKENI ASSPLEDFGNEQF 2 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 580) KGHSH LQKENI ASSLPEGFDNEQF 2 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 581) KGHSH LQKENI ASPLPEGFGNEQF 2 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 582) KGHSH LQKENI ASLPPEGFGNEQF 2 (SEQ ID NO: 38) (SEQ ID NO: 81) (SEQ ID NO: 568) DFQATT SNEGSKA SVWTDSDTQY 2 (SEQ ID NO: 39) (SEQ ID NO: 89) (SEQ ID NO: 583) ASSGPSGQPQH 2 (SEQ ID NO: 584) ASLSSPPRDPWRLIHPS 2 (SEQ ID NO: 585) TCRalpha NYSPAY IRENEKE APYTGRRALT 60408 Donor Y (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 223) (GV10.3) AASGANTNKVV 464 (SEQ ID NO: 586) NYSPAY IRENEKE APYAGRRALT 169 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 587) NYSPAY IRENEKE APCTGRRALT 107 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 588) NYSPAY IRENEKE TPYTGRRALT 61 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 589) NYSPAY IRENEKE VPYTGRRALT 59 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 590) NYSPAY IRENEKE APYMGRRALT 57 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 591) NYSPAY IRENEKE APYTGRRAL 39 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 592) SIFNT LYKAGEL AGQDQDSGYALN 36 (SEQ ID NO: 20) (SEQ ID NO: 84) (SEQ ID NO: 593) NYSPAY IRENEKE APTRAGEHL 16 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 594) IFSNGE AASEGNYNVLY 15 (SEQ ID NO: 85) (SEQ ID NO: 386) DSVNN IPSGT AANSNDYKLS 9 (SEQ ID NO: 6) (SEQ ID NO: 66) (SEQ ID NO: 595) NYSPAY IRENEKE APYPGRRALT 7 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 596) NYSPAY IRENEKE APYKGRRALT 7 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 597) NYSPAY IRENEKE APYSGRRALT 6 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 598) NYSPAY IRENEKE ALLDSGGGADGLT 6 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 599) NYSPAY IRENEKE GPYTGRRALT 5 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 600) AASGANTNKAV 5 (SEQ ID NO: 601) NYSPAY IRENEKE APSTGRRALT 4 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 602) NYSPAY IRENEKE AP*TGRRALT 4 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 603) SSVSVY YLSGSTLV AVSAITQGGSEKLV 3 (SEQ ID NO: 7) (SEQ ID NO: 67) (SEQ ID NO: 604) NYSPAY IRENEKE SPYTGRRALT 3 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 605) NYSPAY IRENEKE PPYTGRRALT 3 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 606) NYSPAY IRENEKE EPYTGRRALT 3 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 607) NYSPAY IRENEKE APYTAGEHL 3 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 608) DSSSTY IFSNMDM AGPGG*QL*ID 3 (SEQ ID NO: 64) (SEQ ID NO: 86) (SEQ ID NO: 609)

NYSPAY IRENEKE APSRAGEHL 2 (SEQ ID NO: 16) (SEQ ID NO: 76) (SEQ ID NO: 610) DSSSTY IFSNMDM AERYNTDKLI 2 (SEQ ID NO: 64) (SEQ ID NO: 86) (SEQ ID NO: 611) AASGANTNKFV 2 (SEQ ID NO: 612) TCRbeta SGHAT FQDESV ASSLGQGNEAF 72824 Donor Y (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 613) (GV10.3) MNHEY SVGAGI ASSLYFGQPQH 36151 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 537) DFQATT SNEGSKA SAREGSGNEQF 395 (SEQ ID NO: 39) (SEQ ID NO: 89) (SEQ ID NO: 614) SGHAT FQDESV ASSLGQGDEAF 276 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 615) SGHAT FQDESV ASSLGRGNEAF 263 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 616) SGHAT FQDESV ASSLGQGSEAF 247 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 617) SGHAT FQDESV ASSLGQGNGAF 243 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 618) SGHAT FQDESV ASSSGQGNEAF 202 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 619) MNHEY SVGAGI ASSLYLGQPQH 132 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 620) SQVTM ANQGSEA SASSGSTDTQY 125 (SEQ ID NO: 36) (SEQ ID NO: 90) (SEQ ID NO: 621) MNHEY SVGAGI ASSLYFGRPQH 114 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 622) MNHEY SVGAGI ASSLYSGQPQH 110 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 623) MNHEY SVGAGI ASGLYFGQPQH 107 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 624) MNHEY SVGAGI ASSLYFGQPS 76 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 625) SGHAT FQDESV ASSLGQGNKAF 73 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 626) MNHEY SVGAGI ASSLCFGQPQH 68 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 627) SQVTM ANQGSEA SVAGTYSYNEQF 66 (SEQ ID NO: 36) (SEQ ID NO: 90) (SEQ ID NO: 628) DFQATT SNEGSKA SARDQIREQF 63 (SEQ ID NO: 39) (SEQ ID NO: 89) (SEQ ID NO: 629) MNHEY SVGEGT ASSDDPRESGANVLT 51 (SEQ ID NO: 42) (SEQ ID NO: 91) (SEQ ID NO: 630) SGHAT FQDESV ASNLGQGNEAF 45 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 631) SGHAT FQDESV ASSLDRGMKL 44 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 632) SQVTM ANQGSEA SVEGTGGLNEQF 41 (SEQ ID NO: 36) (SEQ ID NO: 90) (SEQ ID NO: 633) SGHAT FQDESV ASSLGQGMKL 39 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 634) MNHNS SASEGT ASSLGWRGNSYEQY 39 (SEQ ID NO: 53) (SEQ ID NO: 92) (SEQ ID NO: 635) SGHAT FQDESV ASS*GQGNEAF 34 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 636) MNHEY SVGAGI ASNLYFGQPQH 33 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 637) MNHEY SVGAGI ASSLYFGQPQ 31 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 638) MNHEY SVGAGI ASSLYFG*PQH 30 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 639) SGHAT FQNNGV ASSLVSGGNEQ 29 (SEQ ID NO: 46) (SEQ ID NO: 108) (SEQ ID NO: 640) MNHEY SVGAGI ASSLYFGQSQH 29 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 641) GTSNPN SVGIG AWEEGEAF 29 (SEQ ID NO: 40) (SEQ ID NO: 104) (SEQ ID NO: 642) SGHAT FQDESV ASSLGQGNEA 27 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 643) SGHAT FQDESV ASSLGQGNVAF 26 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 644) SGHAT FQDESV ASSLGQGKEAF 26 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 645) SQVTM ANQGSEA SVDLGWEQY 25 (SEQ ID NO: 36) (SEQ ID NO: 90) (SEQ ID NO: 646) KGHDR SFDVKD ATSDLTGGNEQF 24 (SEQ ID NO: 65) (SEQ ID NO: 127) (SEQ ID NO: 647) SQVTM ANQGSEA SVELAGEADTQY 19 (SEQ ID NO: 36) (SEQ ID NO: 90) (SEQ ID NO: 648) MNHEY SVGAGI AAVYTLGSPS 18 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 649) SGHAT FQDESV ASSMGQGNEAF 17 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 650) MNHEY SVGAGI ASSYTLGSPS 17 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 651) SGHAT FQDESV ASSLGQGNDAF 16 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 652) MNHEY SVGAGI ASSLYFGSPS 16 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 653) DFQATT SNEGSKA SASSGTSGRLYNEQF 16 (SEQ ID NO: 39) (SEQ ID NO: 89) (SEQ ID NO: 654) SGHAT FQDESV AAAWDRGMKL 15 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 655) SGHAT FQDESV ASSWGQGNEAF 13 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 656) SGHAT FQDESV ASSVGQGNEAF 13 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 657) SEHNR FQNEAQ ASSLTLQETQY 13 (SEQ ID NO: 56) (SEQ ID NO: 117) (SEQ ID NO: 658) SQVTM ANQGSEA SVGTSGYEQY 12 (SEQ ID NO: 36) (SEQ ID NO: 90) (SEQ ID NO: 659) MNHEY SVGAGI ASSYFAGPYEQY 11 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 660) GTSNPN SVGIG AWSEGVGNQPQH 11 (SEQ ID NO: 40) (SEQ ID NO: 104) (SEQ ID NO: 661) MNHEY SVGAGI ASS*YFGQPQH 7 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 662) SGHAT FQDESV ASSLGQGNAAF 6 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 663) SGHAT FQDESV ASILGQGNEAF 6 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 664) SEHNR FQNEAQ ASSLVGAQGLAGTNNYEQ 6 (SEQ ID NO: 56) (SEQ ID NO: 117) Y (SEQ ID NO: 665) MNHEY SVGAGI ASSLYFGPPQH 6 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 666) MNHEY SVGAGI ASSLYFGLPQH 6 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 667) MNHEY SVGAGI ASSLYFGHPQH 6 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 668) MNHEY SVGAGI ASSIYFGQPQH 6 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 669) MNHEY SVGAGI ASILYFGQPQH 6 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 670) MDHEN SYDVKM ASSPGSAYNEQF 6 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 671) ENHRY SYGVKD AISESLAGGYNEQF 6 (SEQ ID NO: 48) (SEQ ID NO: 110) (SEQ ID NO: 672) SGHAT FQDESV ASRLGQGNEAF 5 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 673) MNHEY SMNVEV ASSPTLGVDTQY 5 (SEQ ID NO: 42) (SEQ ID NO: 115) (SEQ ID NO: 674) ASSVDGGEQPQH 5 (SEQ ID NO: 675) SGHDN FVKESK ASSQYVEQY 4 (SEQ ID NO: 41) (SEQ ID NO: 105) (SEQ ID NO: 676) MNHEY SVGAGI ASSQGSDEQY 4 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 677) MDHEN SYDVKM ASSLTGHREAYNEQF 4 (SEQ ID NO: 52) (SEQ ID NO: 93) (SEQ ID NO: 678) WSHSY SAAADI ASSSTGGTSYGYT 3 (SEQ ID NO: 49) (SEQ ID NO: 111) (SEQ ID NO: 679) SGHAT FQDESV ASSLGQGTEAF 3 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 680) SGHAT FQDESV ASSLGQGN*AF 3 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 681) SGHAT FQDESV ASSLGQGN 3 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 682) SGHAT FQDESV ASSLGQGDGAF 3 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 683) SEHNR FQNEAQ ASSPRVPGQGTAGNTIY 3 (SEQ ID NO: 56) (SEQ ID NO: 117) (SEQ ID NO: 684) SEHNR FQNEAQ ASSLSVGSGELF 3 (SEQ ID NO: 56) (SEQ ID NO: 117) (SEQ ID NO: 685) MNHEY SVGAGI ASSVYFGQPQH 3 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 686) MNHEY SVGAGI ASSLYFGQTQH 3 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 687) MNHEY SVGAGI ASSLYFGQAQH 3 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 688) MNHEY SVGAGI ASSLYFGKPQH 3 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 689) MNHEY SVGAGI ASRLYFGQPQH 3 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 690) MNHEY SMNVEV ASSLYWVDTQY 3 (SEQ ID NO: 42) (SEQ ID NO: 115) (SEQ ID NO: 691)

MNHEY SMNVEV ASSLAYTSTEAF 3 (SEQ ID NO: 42) (SEQ ID NO: 115) (SEQ ID NO: 692) MNHEY SMDVEV ASSLYQGPNEQF 3 (SEQ ID NO: 42) (SEQ ID NO: 115) (SEQ ID NO: 693) LNHDA SQIVND ASSFRQWAGGGTDTQY 3 (SEQ ID NO: 47) (SEQ ID NO: 109) (SEQ ID NO: 694) SGHRS YFSETQ ASSLVQGTWYEQY 2 (SEQ ID NO: 59) (SEQ ID NO: 124) (SEQ ID NO: 695) SGHAT FQNNGV ASSLVGGAYNEQF 2 (SEQ ID NO: 46) (SEQ ID NO: 108) (SEQ ID NO: 696) SGHAT FQDESV ASSWDRGMKL 2 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 697) SGHAT FQDESV ASSLVSGGNEQF 2 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 698) SGHAT FQDESV ASSLGQGNQAF 2 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 699) SGHAT FQDESV ASSLGQGNEV 2 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 700) SGHAT FQDESV ASSLGQGDKAF 2 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 701) SGHAT FQDESV ASGSGQGNEAF 2 (SEQ ID NO: 46) (SEQ ID NO: 87) (SEQ ID NO: 702) PRHDT FYEKMQ ASSSLLASGLHTQY 2 (SEQ ID NO: 61) (SEQ ID NO: 126) (SEQ ID NO: 703) MNHNS SASEGT ASSPGWRGNSYEQY 2 (SEQ ID NO: 53) (SEQ ID NO: 92) (SEQ ID NO: 704) MNHEY SVGAGI ASSLYSGQPS 2 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 705) MNHEY SVGAGI ASSLYFGEPQH 2 (SEQ ID NO: 42) (SEQ ID NO: 88) (SEQ ID NO: 706) LGHDT FNNKEL ASSQLGQGAGEQY 2 (SEQ ID NO: 51) (SEQ ID NO: 128) (SEQ ID NO: 707) DFQATT SNEGSRA SARGGSGNEQF 2 (SEQ ID NO: 39) (SEQ ID NO: 129) (SEQ ID NO: 708) DFQATT SNEGSKA SAREGSGDEQF 2 (SEQ ID NO: 39) (SEQ ID NO: 89) (SEQ ID NO: 709)

[0117] We synthesize peptides and test their immunogenicity in vitro by peptide-binding assays. Using positive and negative controls, we select a set of peptides containing EGFR or Ras mutations that can be presented with certain HLA alleles and treat the patients' tumor cells in vitro. The clonal cells that respond to these peptides are expanded. Subsequently, we use fluorescence-activated cell sorting to isolate CD8+ T cells from the treated and expanded human cells. The CTLs that responded to peptide stimulation are then genetically induced to yield iPS cells. These iPS cells are cloned and redifferentiated to T-iPS cells with incorporation of the iCas9 safety switch. Further, these rejCTL cells are expanded in vitro and used to treat xenografts in mice.

Example 5

Safeguard Technology

[0118] The tumorigenic potential of undifferentiated iPSCs is a safety concern that must be addressed before iPS cell-based therapies can be routinely used in clinical settings. Using a mouse model, we recently established a way to manipulate a naturally existing suicide pathway to control whether such cells and their progeny live or die. We found that introducing into the cytotoxic T cells a gene encoding a protein called inducible caspase-9, or iC9, permitted us to trigger these cells, and not others, to die throughout the body by activating iC9 with a specific chemical, CID. These engineered T cells still recognize the same antigens, and are just as effective against cancer tumors as are their unmodified peers.sup.1. But they can be quickly eliminated with a simple treatment. This is the first time that a "safeguard system" has been incorporated into in vivo cell-based therapy.

[0119] We use this safeguard technology to generate rejuvenated T-iPS cells from lung cancer patients' tissue, blood, or malignant effusion fluid that contain lung cancer specific antigens. In particular, we use specific antigens of the HLA-specific peptide sequences containing alterations in the EGFR protein around mutations (described above). We select clonal T cells that react with these antigens and reprogram them to monoclonal TCR-expressing T-iPSCs with rejuvenated progeny (rejCTLs). We assay the variance of antigen reactivity during the processes of TiPS generation and T-cell redifferentiation by demonstrating genomic rearrangements in TCR genes. Furthermore, these rejCTLs are infused into mice that harbor lung cancer xenografts to determine the treatment effects.

Example 6

Methods

Statistics

[0120] Statistical analyses is performed using Excel, Prism (Graphpad Software, La Jolla, Calif.), and Statcel 2 (OMS Publishing, Saitama, Japan) programs, applying ANOVA or a paired-sample Student's t-test, with P<0.05 indicative of significance.

Peptide-Binding Assay

[0121] After incubation in culture medium at 26.degree. C. overnight, T2 cells are washed with PBS and suspended in 1 ml Opti-MEM (Invitrogen Life Technologies, Carlsbad, Calif.) with peptide (100 .mu.g/ml), followed by incubation at 26.degree. C. for 3 h and then at 37.degree. C. for 2.5 h. After washing with PBS, HLA expression is measured using a BD FACSCanto II flow cytometer (BD Biosciences, San Jose, Calif.) using a FITC-conjugated HLA-specific monoclonal antibody. Mean fluorescence intensity is analyzed using FlowJo software.

PBMC Collection and Lung Cancer Tissue Collection

[0122] Peripheral blood samples will be collected from lung cancer patients. PBMCs are isolated by density centrifugation and stored frozen in liquid nitrogen until use. Lung cancer tissue is dissociated into primary cancer cells using an established cell isolation protocol with enzymatic digestion to yield single cells. The mixture of cancer cells is then cultured in RPMI-1640 supplemented with 10% FBS.

Generation of DCs

[0123] CD14+ cells are isolated from PBMCs using CD14 microbeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Immature dendritic cells (DCs) will be generated from CD14+ cells using IL-4 (10 ng/ml; PeproTech, Rocky Hill, N.J.) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (10 ng/ml) in RPMI-1640 supplemented with 10% FBS. Maturation of DCs will be induced by prostaglandin E2 (PGE2) (1 .mu.g/ml) and tumor necrosis factor-.alpha. (TNF-.alpha.) (10 ng/ml; PeproTech).

Induction of Peptide-Specific CTLs

[0124] CD8+ T cells (2.times.10.sup.6 cells/well) will be stimulated with peptide-pulsed (10 .mu.g/ml) 100-Gy-irradiated autologous mature DCs (1.times.105 cells/well) in RPMI-1640 containing 10% heat-inactivated human AB serum. After 1 week, these cells will be stimulated twice weekly with peptide-pulsed (10 .mu.g/ml) 200-Gyirradiated aAPC-A2 cells (1.times.10.sup.5 cells/well). Supplementation with 10 IU/ml IL-2 and 10 ng/ml IL-15 (PeproTech) are performed at 3- to 4-day intervals between stimulations.

IFN-.gamma.ELISPOT Assay

[0125] Specific secretion of interferon-y (IFN-.gamma.) from human CTLs in response to stimulator cells are assayed using the IFN-.gamma. enzyme-linked immuno spot (ELISPOT) kit (BD Biosciences, San Jose, Calif.), according to the manufacturer's instructions. Stimulator cells are pulsed with peptide for 2 h at room temperature and then washed three times. Responder cells will be incubated with stimulator cells for 20 h. The resulting spots are counted.

Cytotoxicity Assay

[0126] Cytotoxic capacity are analyzed using the Terascan VPC system (Minerva Tech, Canada). The CTL line is used as the effector cell type. Target cells are labeled in calcein-AM solution for 30 min at 37.degree. C. The labeled cells will then be co-cultured with the effector cells for 4-6 h. Fluorescence intensity will be measured before and after the culture period, and specific cytotoxic activity will be calculated using the following formula: % cytotoxicity={1-[(average fluorescence of the sample wells-average fluorescence of the maximal release control wells)/(average fluorescence of the minimal release control wells-average fluorescence of the maximal release control wells)]}.times.100.

Preparation of T-Cells, Infection, and T-iPS Generation

[0127] T cells will be isolated by gating the CD3+CD56- population to avoid contamination by natural killer T cells. T-cell subsets will be separated, using additional gating strategies, into CD4 (CD4+CD8-) and CD8 (CD4CD8+) cohorts. CD4 and/or CD8 cells are further classed as naive (CD45RA+CD62L+), central memory (CD45RA+CD62L-), effector memory (CD45RA-CD62L-), or terminal effector (CD45RA-CD62L+). Sorted cells, initially cultured in Roswell Park Memorial Institute RPMImedium (GIBCO-Invitrogen, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (GIBCO-Invitrogen), 100 U/ml penicillin, 100 ng/ml streptomycin, 2 mM L-glutamine, and 20 ng/ml human interleukin 2 (hIL-2; Novartis Vaccines & Diagnostics, Emeryville, Calif.), are activated by anti-CD3/CD28-conjugated magnetic beads (Dynabeads.RTM. ClinExVivo.TM. CD3/CD28; Invitrogen) at a 3:1 bead:T cell ratio. We define the date of activation as day 0 in the process of T-iPS cell generation.

[0128] In some experiments, magnetically captured CD3+ cells are separated from PBMNCs and stimulated concurrently with anti-CD3/CD28-conjugated magnetic beads. At days 6 and 7, the cells are infected with sendai virus vector carrying iPS-reprogramming factors. Medium for primary T-cell culture are changed every day. At day 8, infected cells are collected and transferred onto irradiated MEF layers at 3.times.10.sup.5 cells per 6-cm dish. For 4 days thereafter, half-volumes of culture medium are daily replaced with Dulbecco's modified Eagle medium/F12 medium supplemented with 20% Knockout Serum Replacement (GIBCO-Invitrogen), 200 .mu.M L-glutamine (Invitrogen), 1% non-essential amino acids, 10 .mu.M 2-mercaptoethanol, and 5 ng/ml b-FGF as described ("human iPSC medium"). VPA are added at 0.5 mM to human iPS medium before picking up iPSC colonies. At day 12, the entire volume of medium is changed to human iPS medium containing VPA. When human ES/iPS-like colonies become identifiable, around day 21, they are mechanically isolated and dissociated into small clamps by pipetting, with reseeding onto fresh MEF layers. Human ES/iPS-like clones are passed onto new MEF layers every 6 days using trypsin solution (0.25% trypsin, 1 mM CaCl.sub.2), and 20% Knockout Serum Replacement in PBS).

Karyotyping

[0129] Chromosomal G-band analyses are conducted in routine fashion (Nihon Gene Research Laboratories, Miyagi, Japan).

Alkaline Phosphatase Staining and Immunocytochemistry

[0130] Human iPS-like colonies fixed in ice-cold fixative solution (90% methanol, 10% formaldehyde) will bestained using a kit (Vector Laboratories, Burlingame, Calif.) according to manufacturer's instructions. For immunocytochemical staining, human iPS-like colonies fixed in 5% paraformaldehyde are permeabilized with 0.1% Triton X-100. The pretreated colonies are incubated first with primary antibodies (PE-conjugated anti-SSEA-4, 1:50, FAB1435P, R&D Systems, Minneapolis, Minn.; anti-TRA-160, 1:100, MAB4360, Millipore, Billerica; or anti-TRA-1-81, 1:100, MAB4381, Millipore). The secondary antibody used for TRA-1-60 and TRA-1-81 Will be Alexa Fluor 488-conjugated goat anti-mouse antibody (1:500; A11029, Molecular Probes-Invitrogen). Nuclei are counterstained with DAPI; 1:1000 (Roche Diagnostics, Indianapolis, Ind.). Photographs will be taken using a fluorescence microscope.

Teratoma Formation

[0131] Human iPS-like colonies are clumped and injected (1.0.times.10.sup.6 cells/mouse) into the medulla of the left testis of NOD-SCID mice. Eight weeks after injection, tumors formed in the testis are resected, fixed in 5% paraformaldehyde, and embedded in paraffin. Sections are stained with hematoxylin/eosin technique and examined by light microscopy for evidence of tri-lineage germ layer differentiation.

Pluripotent Genes and T-Cell-Related Genes Expression Analysis

[0132] Using an RNeasy mini kit (Qiagen, Hilden, Germany), total RNA will be extracted from iPS cells (about 50 days after cloning), their progeny cells, and freshly isolated peripheral-blood CD3 T-cells. Total RNA (1 .mu.g) are reverse transcribed with a PrimeScript III cDNA Synthesis Kit (Invitrogen). PCRs are performed using ExTaq HS (Takara) at 30 cycles for housekeeping genes (GAPDH or ACTB) and at 35 cycles for all pluripotent or T-cell related genes.

Detecting TCR Rearrangement in Genomic DNA of T-iPS Cells

[0133] Genomic DNA are extracted from approximately 5.times.10.sup.6 T-iPS cells using QIAamp DNA kits (Qiagen). Extracted DNA (40 ng) are used in each PCR to detect TCRG, TCRB and TCRA gene rearrangements. PCRs for detecting TCRG rearrangement are performed. The V, D, and J segments involved in assembled TCRA or TCRB are identified by comparison with published sequences and with the ImMunoGeneTics (IMGT) database (cines.fr/), as well as by using web tools such as v-quest. Gene-segment nomenclature follows IMGT usage.

Induction of T-Lineage Cells from T-iPS Cells

[0134] Briefly, iPS cells are co-cultured on an irradiated OP9 layer for 10 to 14 days in DMEM medium without cytokines. Floating cells packed and transferred onto OP9-DL1 layers (day 0), are co-cultured in .alpha.MEM-based medium supplemented with 10 ng/ml of hIL-7 and hFlt-3L for up to day 28. The culture medium is changed every 3 days. T-lineage cells, floating above OP9-DL1 layers and expressing CD45, CD3, and TCR, are sorted by flow cytometry weekly and gene expression analyses will be carried out.

Antitumor Activity in In Vivo Model

[0135] Treatment efficacy is evaluated in SCID mice engrafted with patient NSCLCs. To evaluate the antitumor effects of rejT-iC9-CTLs in SCID mice engrafted with lung cancer, tumor growth is monitored using a bioluminescence system. Once a progressive increase of bioluminescence occurs, mice are treated intraperitoneally with 3 once weekly doses of rejT-iC9-CTL and control CTLs (10.times.10.sup.6 CTL/mouse). Tumor burden is monitored by the Xenogen-IVIS imaging system. Mice are injected intraperitoneally with d-luciferin (150 mg/kg) and light output is analyzed using the Xenogen Living Image Software Version 2.50 (Xenogen, Alameda, Calif.).

In Vivo Elimination of iC9-iPSC-Derived CTLs

[0136] To examine whether iC9-iPSC-derived CTLs can be eliminated by this iC9/CID safeguard system in vivo, SCID mice engrafted with lung cancer tissues are treated with CID (50 .mu.g i.p. daily for three successive days (day 2-day 4). Comparison mice will not receive CID.

Detection of rejT-iC9-CTLs in vivo

[0137] SCID mice inoculated intraperitoneally with iC9-iPSC-derived CTLs labeled with GFP/FFluc are treated with 10.times.10.sup.6 rejT-iC9- cells on day 0 and day 7. After rejT-iC9- cells are detected in peripheral blood (around 8 days after first rejT-iC9 administration), the mice receive intraperitoneally injected CID, at 50 ug/mouse, for three successive days. Control mice I receive three doses of PBS. Flow cytometry of peripheral blood is used to identify rejT-iC9-cells (expressing mCherry).

Establishment of NSCLC Cell Lines and Tissues Engraftable into Immunodeficient Mice

[0138] Lung cancer tissue samples are collected from surgically resected tumors. We have an established lung cancer tissue dissociation protocol that can be used to precede primary culture. Whole blood is processed for T cell culture and used for the establishment of NSCLC-specific CTLs. Patient tumor tissue or cells in malignant effusions from cancer patients are used to establish xenografts in SCID mice. Briefly, patient's tumor chunks or malignant-effusion cells are transplanted into SCID mice. Tumor size is measured in these mice to assess the progress of lung cancer in this in vivo model. SCID mice successfully engrafted with tumor will be used for further treatments.

REFERENCES

[0139] 1. Nishimura, T., Kaneko, S., Kawana-Tachikawa, A., Tajima, Y., Goto, H., Zhu, D., NakayamaHosoya, K., Iriguchi, S., Uemura, Y., Shimizu, T., et al. 2013. Generation of rejuvenated antigen-specific T cells by reprogramming to pluripotency and redifferentiation. Cell Stem Cell 12:114-126.

[0140] 2. Vodyanik, M. A., Bork, J. A., Thomson, J. A., and Slukvin, I I. 2005. Human embryonic stem cellderived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential. Blood 105:617-626.

[0141] 3. Takayama, N., Nishikii, H., Usui, J., Tsukui, H., Sawaguchi, A., Hiroyama, T., Eto, K., and Nakauchi, H. 2008. Generation of functional platelets from human embryonic stem cells in vitro via ES-sacs, VEGF-promoted structures that concentrate hematopoietic progenitors. Blood 111:5298-5306.

[0142] 4. Schmitt, T. M., and Zuniga-Pflucker, J. C. 2002. Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro. Immunity 17:749-756.

[0143] 5. La Motte-Mohs, R. N., Herer, E., and Zuniga-Pflucker, J. C. 2005. Induction of T-cell development from human cord blood hematopoietic stem cells by Delta-like 1 in vitro. Blood 105:1431-1439.

[0144] 6. Vizcardo, R., Masuda, K., Yamada, D., Ikawa, T., Shimizu, K., Fujii, S., Koseki, H., and Kawamoto, H. 2013. Regeneration of human tumor antigen-specific T cells from iPSCs derived from mature CD8(+) T cells. Cell Stem Cell 12:31-36.

[0145] 7. Chen, G., Gulbranson, D. R., Hou, Z., Bolin, J. M., Ruotti, V., Probasco, M. D., Smuga-Otto, K., Howden, S. E., Diol, N. R., Propson, N. E., et al. 2011. Chemically defined conditions for human iPSC derivation and culture. Nat Methods 8:424-429.

[0146] 8. Ando M, Nishimura T, Yamazaki S, Yamaguchi T, Kawana-Tachikawa A, Hayama T, Nakauchi Y, Ando J, Ota Y, Takahashi S, Nishimura K, Ohtaka M, Nakanishi M, Miles J J, Burrows S R, Brenner M K, Nakauchi H. A Safeguard System for Induced Pluripotent Stem Cell-Derived Rejuvenated T Cell Therapy 2015 Oct. 13; 5(4):597-608. Stem Cell Report

[0147] 9. Yagyu S, Hoyos V, Del Bufalo F, Brenner M K 2015 An Inducible Caspase-9 Suicide Gene to Improve the Safety of Therapy Using Human Induced Pluripotent Stem Cells. Mol Ther. 2015 September; 23(9):1475-85

[0148] 10. Lefranc, M. P. 2003. IMGT databases, web resources and tools for immunoglobulin and T cell receptor sequence analysis, http://imgt.cines.fr. Leukemia 17:260-266.

[0149] 11. Yamada T, Azuma K, Muta E, Kim J, Sugawara S, Zhang G L, Matsueda S, KasamaKawaguchi Y, Yamashita Y, Yamashita T, Nishio K, Itoh K, Hoshino T, Sasada T. EGFR T790M mutation as a possible target for immunotherapy; identification of HLA-A*0201restricted T cell epitopes derived from the EGFR T790M mutation. PLoS One. 2013 Nov. 5; 8(11)

[0150] 12. Ofuji K, Tada Y, Yoshikawa T, Shimomura M, Yoshimura M, Saito K, Nakamoto Y, Nakatsura T. A peptide antigen derived from EGFR T790M is immunogenic in non-small cell lung cancer. Int J Oncol. 2015 February; 46(2):497-504

[0151] 13. NCCN Clinical Practice Guidelines in Oncology.TM.. Non-small cell lung cancer. v 2.2010. Available at: http://www.nccn.org/professionals/physician_gls/f_guidelines.asp. Accessed Jul. 22, 2010

[0152] 14. West H, Lilenbaum R, Harpole D, et al. Molecular analysis-based treatment strategies for the management of non-small cell lung cancer. J Thorac Oncol. 2009; 4:s1029-s1039.

[0153] 15. Jackman D M, Miller V A, Cioffredi L A, et al. Impact of epidermal growth factor receptor and KRAS mutations on clinical outcomes in previously untreated non-small cell lung cancer patients: Results of an online tumor registry of clinical trials. Clin Cancer Res. 2009; 15:5267-5273

[0154] 16. Klebanoff C A, Rosenberg S A, Restifo N P Prospects for gene-engineered T cell immunotherapy for solid cancers. Nat Med. 2016 Jan. 6; 22(1):26-3

[0155] 17. Skora A D, Douglass J, Hwang M S, Tam A J, Blosser R L, Gabelli S B, Cao J, Diaz L A Jr, Papadopoulos N, Kinzler K W, Vogelstein B, Zhou S Generation of MANAbodies specific to HLArestricted epitopes encoded by somatically mutated genes. Proc Natl Acad Sci USA. 2015 Aug. 11; 112(32):9967-72

[0156] 18. Morello A, Sadelain M, Adusumilli P S, Mesothelin-Targeted CARs: Driving T Cells to Solid Tumors Cancer Discov. 2016 February; 6(2):133-46

[0157] 19. Adusumilli P S, Cherkassky L, Villena-Vargas J, Colovos C, Servais E, Plotkin J, Jones D R, Sadelain M. Regional delivery of mesothelin-targeted CAR T cell therapy generates potent and long-lasting CD4-dependent tumor immunity. Sci Transl Med. 2014 Nov. 5; 6(261):261ra151

[0158] 20. Beatty G L, Haas A R, Maus M V, Torigian D A, Soulen M C, Plesa G, Chew A, Zhao Y, Levine B L, Albelda S M, Kalos M, June C H, Mesothelin-specific chimeric antigen receptor mRNA-engineered T cells induce anti-tumor activity in solid malignancies Cancer Immunol Res. 2014 February; 2(2):11220

[0159] 21. Gao X, Le X, Costa D B, The safety and efficacy of osimertinib for the treatment of EGFR T790M mutation positive non-small-cell lung cancer Expert Rev Anticancer Ther. 2016 Mar. 4

[0160] 22. Noda S, Kanda S Addressing epidermal growth factor receptor tyrosine kinase inhibitor resistance in non-small cell lung cancer Expert Rev Respir Med. 2016 Mar. 9

[0161] 23. van der Wekken A J, Saber A, Hiltermann T J, Kok K, van den Berg A, Groen H J. Resistance mechanisms after tyrosine kinase inhibitors afatinib and crizotinib in non-small cell lung cancer, a review of the literature Crit Rev Oncol Hematol. 2016 Jan. 25

[0162] 24. Villadolid J, Ersek J L, Fong M K, Sirianno L, Story E S Management of hyperglycemia from epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) targeting T790Mmediated resistance Transl Lung Cancer Res. 2015 October; 4(5):576-83

[0163] 25. Black R C, Khurshid H NSCLC: An Update of Driver Mutations, Their Role in Pathogenesis and Clinical Significance R I Med J (2013). 2015 Oct. 1; 98(10):25-8

[0164] 26. Berman A T, Simone C B 2nd Immunotherapy in locally-advanced non-small cell lung cancer: releasing the brakes on consolidation? Transl Lung Cancer Res. 2016 February; 5(1):138-42

[0165] 27. Ahmad S M, Borch T H, Hansen M, Andersen M H PD-L1-specific T cells Cancer Immunol Immunother. 2016 Jan. 2

[0166] 28. Zhukovsky E A, Morse R J, Maus M V Bispecific antibodies and CARs: generalized immunotherapeutics harnessing T cell redirection Curr Opin Immunol. 2016 Mar. 7; 40:24-35

[0167] 29. Simon S, Vignard V, Florenceau L, Dreno B, Khammari A, Lang F, Labarriere N PD-1 expression conditions T cell avidity within an antigen-specific repertoire Oncoimmunology. 2015 Oct. 29; 5(1)

[0168] 30. Hasegawa K, Tanaka S, Fujiki F, Morimoto S, Nakajima H, Tatsumi N, Nakata J, Takashima S, Nishida S, Tsuboi A, Oka Y, Oji Y, Kumanogoh A, Sugiyama H, Hosen N An Immunocompetent Mouse Model for MLL/AF9 Leukemia Reveals the Potential of Spontaneous Cytotoxic T-Cell Response to an Antigen Expressed in Leukemia Cells PLoS One. 2015 Dec. 11; 10(12)

[0169] 31. Zou W, Wolchok J D, Chen L PD-L1 (B7-H1) and PD-1 pathway blockade for cancer therapy: Mechanisms, response biomarkers, and combinations Sci Transl Med. 2016 Mar. 2; 8(328):328

[0170] 32. Naidoo J, Schindler K, Querfeld C, Busam K J, Cunningham J, Page D B, Postow M A, Weinstein A, Skripnik Lucas A, Ciccolini K T, Quigley E A, Lesokhin A M, Paik P K, Chaft J E, Segal N H, D'Angelo S P, Dickson M A, Wolchok J D, Lacouture M E Autoimmune Bullous Skin Disorders with Immune Checkpoint Inhibitors Targeting PD-1 and PD-L1 Cancer Immunol Res. 2016 Feb. 29

Sequence CWU 1

1

70919PRTArtificial SequenceCS9.3 EGFR peptide with C797S 1Gln Leu Met Pro Phe Gly Ser Leu Leu1 5211PRTArtificial SequenceCS11.6 EGFR peptide with C797S mutation 2Leu Met Pro Phe Gly Ser Leu Leu Asp Tyr Val1 5 10310PRTArtificial SequenceGD10.3 KRAS peptide with G12D mutation 3Lys Leu Val Val Val Gly Ala Asp Gly Val1 5 10410PRTArtificial SequenceGV10.3 KRAS peptide with G12V mutation 4Lys Leu Val Val Val Gly Ala Val Gly Val1 5 10510PRTArtificial SequenceGC10.3 KRAS peptide with G12C mutation 5Lys Leu Val Val Val Gly Ala Cys Gly Val1 5 1065PRTHomo sapiens 6Asp Ser Val Asn Asn1 576PRTHomo sapiens 7Ser Ser Val Ser Val Tyr1 587PRTHomo sapiens 8Thr Ile Ser Gly Asn Glu Tyr1 596PRTHomo sapiens 9Ser Ser Val Pro Pro Tyr1 5106PRTHomo sapiens 10Val Ser Pro Phe Ser Asn1 5115PRTHomo sapiens 11Thr Ser Ile Asn Asn1 5126PRTHomo sapiens 12Ser Ser Asn Phe Tyr Ala1 5136PRTHomo sapiens 13Asp Arg Gly Ser Gln Ser1 5147PRTHomo sapiens 14Thr Ser Glu Ser Asp Tyr Tyr1 5156PRTHomo sapiens 15Thr Ser Gly Phe Asn Gly1 5166PRTHomo sapiens 16Asn Tyr Ser Pro Ala Tyr1 5177PRTHomo sapiens 17Thr Arg Asp Thr Thr Tyr Tyr1 5187PRTHomo sapiens 18Thr Ser Asp Pro Ser Tyr Gly1 5196PRTHomo sapiens 19Thr Ser Gly Phe Tyr Gly1 5205PRTHomo sapiens 20Ser Ile Phe Asn Thr1 5216PRTHomo sapiens 21Tyr Ser Gly Ser Pro Glu1 5226PRTHomo sapiens 22Ser Ser Tyr Ser Pro Ser1 5236PRTHomo sapiens 23Asn Ser Ala Phe Gln Tyr1 5246PRTHomo sapiens 24Asp Ser Ala Ser Asn Tyr1 5256PRTHomo sapiens 25Asn Ser Ala Ser Asp Tyr1 5266PRTHomo sapiens 26Tyr Gly Gly Thr Val Asn1 5276PRTHomo sapiens 27Asn Thr Ala Phe Asp Tyr1 5287PRTHomo sapiens 28Thr Ser Asp Gln Ser Tyr Gly1 5296PRTHomo sapiens 29Asp Ser Ala Ile Tyr Asn1 5306PRTHomo sapiens 30Tyr Gly Ala Thr Pro Tyr1 5317PRTHomo sapiens 31Thr Ser Glu Asn Asn Tyr Tyr1 5326PRTHomo sapiens 32Asp Arg Val Ser Gln Ser1 5336PRTHomo sapiens 33Ala Thr Gly Tyr Pro Ser1 5345PRTHomo sapiens 34Ser Gly His Asn Thr1 5355PRTHomo sapiens 35Ser Gly His Asp Thr1 5365PRTHomo sapiens 36Ser Gln Val Thr Met1 5375PRTHomo sapiens 37Ser Gly His Asp Tyr1 5385PRTHomo sapiens 38Lys Gly His Ser His1 5396PRTHomo sapiens 39Asp Phe Gln Ala Thr Thr1 5406PRTHomo sapiens 40Gly Thr Ser Asn Pro Asn1 5415PRTHomo sapiens 41Ser Gly His Asp Asn1 5425PRTHomo sapiens 42Met Asn His Glu Tyr1 5435PRTHomo sapiens 43Ser Gly His Val Ser1 5445PRTHomo sapiens 44Ser Asn His Leu Tyr1 5455PRTHomo sapiens 45Leu Asn His Asn Val1 5465PRTHomo sapiens 46Ser Gly His Ala Thr1 5475PRTHomo sapiens 47Leu Asn His Asp Ala1 5485PRTHomo sapiens 48Glu Asn His Arg Tyr1 5495PRTHomo sapiens 49Trp Ser His Ser Tyr1 5505PRTHomo sapiens 50Leu Gly His Asn Thr1 5515PRTHomo sapiens 51Leu Gly His Asp Thr1 5525PRTHoJo virus 52Met Asp His Glu Asn1 5535PRTHomo sapiens 53Met Asn His Asn Ser1 5545PRTHomo sapiens 54Met Gly His Arg Ala1 5555PRTHomo sapiens 55Ser Gly His Tyr Tyr1 5565PRTHomo sapiens 56Ser Glu His Asn Arg1 5575PRTHomo sapiens 57Pro Gly His Asn Thr1 5585PRTHomo sapiens 58Ser Val Phe Ser Ser1 5595PRTHomo sapiens 59Ser Gly His Arg Ser1 5605PRTHomo sapiens 60Met Arg His Asn Ala1 5615PRTHomo sapiens 61Pro Arg His Asp Thr1 5625PRTHomo sapiens 62Met Asp His Glu Tyr1 5635PRTHomo sapiens 63Lys Gly His Ser Arg1 5646PRTHomo sapiens 64Asp Ser Ser Ser Thr Tyr1 5655PRTHomo sapiens 65Lys Gly His Asp Arg1 5665PRTHomo sapiens 66Ile Pro Ser Gly Thr1 5678PRTHomo sapiens 67Tyr Leu Ser Gly Ser Thr Leu Val1 5685PRTHomo sapiens 68Gly Leu Lys Asn Asn1 5698PRTHomo sapiens 69Tyr Thr Ser Ala Ala Thr Leu Val1 5707PRTHomo sapiens 70Met Thr Phe Ser Glu Asn Thr1 5717PRTHomo sapiens 71Ile Arg Ser Asn Glu Arg Glu1 5727PRTHomo sapiens 72Met Thr Leu Asn Gly Asp Glu1 5736PRTHomo sapiens 73Ile Tyr Ser Asn Gly Asp1 5748PRTHomo sapiens 74Gln Glu Ala Tyr Lys Gln Gln Asn1 5756PRTHomo sapiens 75Asn Val Leu Asp Gly Leu1 5767PRTHomo sapiens 76Ile Arg Glu Asn Glu Lys Glu1 5778PRTHomo sapiens 77Arg Asn Ser Phe Asp Glu Gln Asn1 5785PRTHomo sapiens 78Gly Leu Thr Ser Asn1 5797PRTHomo sapiens 79Thr Ile Ser Gly Thr Asp Tyr1 5807PRTHomo sapiens 80Ile Arg Ser Asn Val Gly Glu1 5816PRTHomo sapiens 81Leu Gln Lys Glu Asn Ile1 5828PRTHomo sapiens 82Gln Gly Ser Tyr Asp Gln Gln Asn1 5836PRTHomo sapiens 83Asp Ala Leu Asp Gly Leu1 5847PRTHomo sapiens 84Leu Tyr Lys Ala Gly Glu Leu1 5856PRTHomo sapiens 85Ile Phe Ser Asn Gly Glu1 5867PRTHomo sapiens 86Ile Phe Ser Asn Met Asp Met1 5876PRTHomo sapiens 87Phe Gln Asp Glu Ser Val1 5886PRTHomo sapiens 88Ser Val Gly Ala Gly Ile1 5897PRTHomo sapiens 89Ser Asn Glu Gly Ser Lys Ala1 5907PRTHomo sapiens 90Ala Asn Gln Gly Ser Glu Ala1 5916PRTHomo sapiens 91Ser Val Gly Glu Gly Thr1 5926PRTHomo sapiens 92Ser Ala Ser Glu Gly Thr1 5936PRTHomo sapiens 93Ser Tyr Asp Val Lys Met1 5946PRTHomo sapiens 94Phe Tyr Asn Asn Glu Ile1 5954PRTHomo sapiens 95His Ile Ser Arg1968PRTHomo sapiens 96Tyr Thr Thr Gly Ala Thr Leu Val1 5976PRTHomo sapiens 97Thr Tyr Ser Ser Gly Asn1 5987PRTHomo sapiens 98Ile Arg Ser Asn Met Asp Lys1 5998PRTHomo sapiens 99Tyr Phe Ser Gly Asp Pro Leu Val1 51007PRTHomo sapiens 100Ala Thr Lys Ala Asp Asp Lys1 51016PRTHomo sapiens 101Tyr Tyr Arg Glu Glu Glu1 51026PRTHomo sapiens 102Tyr Tyr Glu Glu Glu Glu1 51036PRTHomo sapiens 103Phe Asn Asn Asn Val Pro1 51045PRTHomo sapiens 104Ser Val Gly Ile Gly1 51056PRTHomo sapiens 105Phe Val Lys Glu Ser Lys1 51066PRTHomo sapiens 106Phe Asn Tyr Glu Ala Gln1 51076PRTHomo sapiens 107Tyr Tyr Asp Lys Asp Phe1 51086PRTHomo sapiens 108Phe Gln Asn Asn Gly Val1 51096PRTHomo sapiens 109Ser Gln Ile Val Asn Asp1 51106PRTHomo sapiens 110Ser Tyr Gly Val Lys Asp1 51116PRTHomo sapiens 111Ser Ala Ala Ala Asp Ile1 51126PRTHomo sapiens 112Phe Arg Asn Arg Ala Pro1 51136PRTHomo sapiens 113Tyr Tyr Gly Glu Glu Glu1 51146PRTHomo sapiens 114Tyr Asn Asn Lys Glu Leu1 51156PRTHomo sapiens 115Ser Met Asn Val Glu Val1 51166PRTHomo sapiens 116Tyr Ser Tyr Glu Lys Leu1 51176PRTHomo sapiens 117Phe Gln Asn Glu Ala Gln1 51187PRTHomo sapiens 118Val Val Thr Gly Gly Glu Val1 51196PRTHomo sapiens 119Ser Gln Val Val Asn Asp1 51206PRTHomo sapiens 120Ile Arg Pro Asp Val Ser1 51218PRTHomo sapiens 121Gln Gly Ser Tyr Asp Glu Gln Asn1 51227PRTHomo sapiens 122Ile Gln Ser Ser Gln Arg Glu1 51238PRTHomo sapiens 123Tyr Phe Ser Gly Asp Thr Leu Val1 51246PRTHomo sapiens 124Tyr Phe Ser Glu Thr Gln1 51256PRTHomo sapiens 125Ser Asn Thr Ala Gly Thr1 51266PRTHomo sapiens 126Phe Tyr Glu Lys Met Gln1 51276PRTHomo sapiens 127Ser Phe Asp Val Lys Asp1 51286PRTHomo sapiens 128Phe Asn Asn Lys Glu Leu1 51297PRTHomo sapiens 129Ser Asn Glu Gly Ser Arg Ala1 513010PRTHomo sapiens 130Ala Val Asp Asn Tyr Gly Gln Asn Phe Val1 5 1013110PRTHomo sapiens 131Ala Val Gly Asn Tyr Gly Gln Asn Phe Val1 5 1013210PRTHomo sapiens 132Ala Val Asp Ser Tyr Gly Gln Asn Phe Val1 5 1013310PRTHomo sapiens 133Ala Ala Asp Asn Tyr Gly Gln Asn Phe Val1 5 1013410PRTHomo sapiens 134Ala Val Asp Asp Tyr Gly Gln Asn Phe Val1 5 1013512PRTHomo sapiens 135Ala Val Thr Phe Thr Gly Gly Gly Asn Lys Leu Thr1 5 1013610PRTHomo sapiens 136Ala Val Asn Asn Tyr Gly Gln Asn Phe Val1 5 1013713PRTHomo sapiens 137Ile Val Asn Trp Gly Ser Asn Ser Gly Tyr Ala Leu Asn1 5 1013810PRTHomo sapiens 138Ala Glu Asp Asn Tyr Gly Gln Asn Phe Val1 5 1013910PRTHomo sapiens 139Ala Val Val Asn Tyr Gly Gln Asn Phe Val1 5 1014010PRTHomo sapiens 140Ala Val Asp Ile Tyr Gly Gln Asn Phe Val1 5 1014115PRTHomo sapiens 141Ala Val Ser Glu Met Asn Tyr Gly Gly Ser Gln Gly Asn Leu Ile1 5 10 151428PRTHomo sapiens 142Ala Val Glu Gly Tyr Lys Leu Ser1 514310PRTHomo sapiens 143Val Ala Met Asn Arg Asp Asp Lys Ile Ile1 5 1014410PRTHomo sapiens 144Ala Val Glu Asn Tyr Gly Gln Asn Phe Val1 5 1014512PRTHomo sapiens 145Ala Thr Val Ser Thr Ser Gly Thr Tyr Lys Tyr Ile1 5 1014611PRTHomo sapiens 146Ala Val Ser Asp Thr Gly Phe Gln Lys Leu Val1 5 1014710PRTHomo sapiens 147Ala Val Asp Tyr Tyr Gly Gln Asn Phe Val1 5 1014810PRTHomo sapiens 148Ala Val Tyr Asn Tyr Gly Gln Asn Phe Val1 5 1014910PRTHomo sapiens 149Ala Gly Asp Asn Tyr Gly Gln Asn Phe Val1 5 1015010PRTHomo sapiens 150Ala Val Asp Thr Tyr Gly Gln Asn Phe Val1 5 1015110PRTHomo sapiens 151Ala Val Ala Asn Tyr Gly Gln Asn Phe Val1 5 1015210PRTHomo sapiens 152Ala Phe Met Ser Gly Tyr Ser Thr Leu Thr1 5 1015313PRTHomo sapiens 153Ala Val Asn Leu Gly Gly Gly Gly Ala Asp Gly Leu Thr1 5 1015414PRTHomo sapiens 154Ala Val Glu Gly Tyr Ser Gly Ala Gly Ser Tyr Gln Leu Thr1 5 1015510PRTHomo sapiens 155Ala Val Asp His Tyr Gly Gln Asn Phe Val1 5 1015610PRTHomo sapiens 156Ala Val Glu Pro His Asn Ala Arg Leu Met1 5 1015710PRTHomo sapiens 157Ala Val Asp Lys Tyr Gly Gln Asn Phe Val1 5 1015812PRTHomo sapiens 158Ala Tyr Arg Ser Ala Val Thr Gly Asn Gln Phe Tyr1 5 1015910PRTHomo sapiens 159Ala Val His Asn Tyr Gly Gln Asn Phe Val1 5 1016012PRTHomo sapiens 160Ala Val Asp Leu Asn Ser Gly Tyr Ser Thr Leu Thr1 5 1016111PRTHomo sapiens 161Ala Val Ser Asp Pro Gly Asp Glu Lys Leu Thr1 5 1016213PRTHomo sapiens 162Ala Thr Val Gln Asn Thr Gly Thr Ala Ser Lys Leu Thr1 5 101639PRTHomo sapiens 163Ala Leu Gly Thr Glu Met Thr Arg Ser1 516412PRTHomo sapiens 164Ala Leu Ser Asp Ser Glu Gly Ala Gln Lys Leu Val1 5 101658PRTHomo sapiens 165Ala Val Asp Gly Gln Lys Leu Leu1 516611PRTHomo sapiens 166Ala Met Arg Glu Gly Gly Asp Asp Lys Ile Ile1 5 1016714PRTHomo sapiens 167Ile Val Arg Val Ala Ser Gly Gly Gly Ala Asp Gly Leu Thr1 5 101688PRTHomo sapiens 168Ala Leu Arg Arg Leu Gln Asn Tyr1 516912PRTHomo sapiens 169Ala Val Leu Pro Gln Gly Gly Ser Glu Lys Leu Val1 5 1017010PRTHomo sapiens 170Ala Val Asp Asn Arg Gly Gln Asn Phe Val1 5 101719PRTHomo sapiens 171Ala Leu Tyr Asn Phe Asn Lys Phe Tyr1 517211PRTHomo sapiens 172Ala Gly Gln Leu Thr Leu Ala Thr Gln Ala Asn1 5 1017310PRTHomo sapiens 173Ala Val Asp Ser Cys Gly Gln Asn Phe Val1 5 101749PRTHomo sapiens 174Ala Gly Ile Thr Met Val Arg Ile Leu1 517512PRTHomo sapiens 175Ala Gln Gly Ser Leu Ala Leu Ala Thr Gln Ala Asn1 5 1017612PRTHomo sapiens 176Ala Cys Phe Asn Ser Asn Ser Gly Tyr Ala Leu Asn1 5 1017712PRTHomo sapiens 177Ala Met Arg Ala Ser Gly Gly Tyr Gln Lys Val Thr1 5 1017813PRTHomo sapiens 178Val Val Ser Arg Ile Met Glu Glu Ala Lys Glu Ile Ser1 5 1017910PRTHomo sapiens 179Ala Val Ser Gly Tyr Asn Asn Asp Met Arg1 5 1018010PRTHomo sapiens 180Ala Val Gly Thr Gly Ala Asn Asn Leu Phe1 5 101818PRTHomo sapiens 181Ala Gly Leu Trp Ser Glu Phe Cys1 51827PRTHomo sapiens 182Ala Ala Ser Ile Met Thr Cys1 518312PRTHomo sapiens 183Ala Met Asp Val Tyr Asn Gln Gly Gly Lys Leu Ile1 5 1018410PRTHomo sapiens 184Val Val Ser Gly Val Gly Gln Asn Phe Val1 5 1018510PRTHomo sapiens 185Ala Val Asp Asn Cys Gly Gln Asn Phe Val1 5 1018610PRTHomo sapiens 186Ala Val Asp Asp His Gly Gln Asn Phe Val1 5 1018710PRTHomo sapiens 187Ala Met Asp Asn Tyr Gly Gln Asn Phe Val1 5 1018812PRTHomo sapiens 188Ala Val Thr Phe Ala Gly Gly Gly Asn Lys Leu Thr1 5 1018912PRTHomo sapiens 189Ala Val Ala Phe Thr Gly Gly Gly Asn Lys Leu Thr1 5 1019010PRTHomo sapiens 190Ala Val Ser Leu Asn Asp Tyr Lys Leu Ser1 5 1019112PRTHomo sapiens 191Ala Glu Ile Ser Tyr Ser Ser Ala Ser Lys Ile Ile1 5 1019210PRTHomo sapiens 192Thr Val Asp Asn Tyr Gly Gln Asn Phe Val1 5 1019310PRTHomo sapiens 193Ala Val Asp Asn Tyr Ser Gln Asn Phe Val1 5 1019410PRTHomo sapiens 194Ala Val Asp Asn His Gly Gln Asn Phe Val1 5 101958PRTHomo sapiens 195Ala Tyr Arg Ser His Asp Met Arg1 519610PRTHomo sapiens 196Ala Tyr Gly Gly Gly Ser Glu Lys Leu Val1 5 1019712PRTHomo sapiens 197Ala Val Thr Ser Thr Gly Gly Gly Asn Lys Leu Thr1 5 1019812PRTHomo sapiens 198Ala Val Thr Leu Thr Gly Gly Gly Asn Lys Leu Thr1 5 1019911PRTHomo sapiens 199Ala Pro Pro Ser Gly Ser Ala Arg Gln Leu Thr1 5 1020010PRTHomo sapiens 200Ala Val Asn Ser Tyr Gly Gln Asn Phe Val1 5 1020110PRTHomo sapiens 201Ala Val Asn Asp Tyr Gly Gln Asn Phe Val1 5 1020210PRTHomo sapiens 202Ala Ala Val Asn Tyr Gly Gln Asn Phe Val1 5 1020310PRTHomo sapiens 203Ala Ala Asn Asn Tyr Gly Gln Asn Phe Val1 5 1020411PRTHomo sapiens 204Ala Val Asn Arg Asn Thr Gly Asn Gln Phe Tyr1 5 1020511PRTHomo sapiens 205Val Val Ser Ala Lys Glu Ala Lys Glu Ile Ser1 5 1020616PRTHomo sapiens 206Val Val Ser Ala Glu Gly Arg Gln Arg Leu Asn Pro Gly Glu Ala Ile1 5 10 1520710PRTHomo sapiens 207Ala Thr Gly Ser Asn Asp Tyr Lys Leu Ser1 5 1020811PRTHomo sapiens 208Ala Thr Asp Gly Arg Gly Ser Tyr Ile Pro Thr1 5 1020911PRTHomo sapiens 209Ala Thr Asp Glu Asp Ser Ser Tyr Lys Leu Ile1 5 1021010PRTHomo sapiens 210Ala Val Ser Asp Ser Asn Tyr Gln Leu Ile1 5 1021113PRTHomo sapiens 211Ala Tyr Arg Ser Ala Gly Gly Ala Thr Asn Lys Leu Ile1 5 1021212PRTHomo sapiens 212Ala Phe Met Lys His Ser Gly Val Asn Asp Met Arg1 5 1021313PRTHomo sapiens 213Ile Val Ser Trp Gly Ser Asn Ser Gly Tyr Ala Leu Asn1 5 102149PRTHomo sapiens 214Ile Cys Ser Gly Asn Thr Pro Leu Val1 521513PRTHomo sapiens 215Ile Ala Asn Trp Gly Ser Asn Ser Gly Tyr Ala Leu Asn1 5 1021612PRTHomo sapiens 216Ala Val Thr Tyr Thr Gly Gly Gly Asn Lys Leu Thr1 5 1021712PRTHomo sapiens 217Ala Val Thr Phe Met Gly Gly Gly Asn Lys Leu Thr1 5 1021812PRTHomo sapiens 218Ala Val Thr Phe Lys Gly Gly Gly Asn Lys Leu Thr1 5 1021910PRTHomo sapiens 219Ala Val Ser Asp Arg Gly Glu Thr Ser Trp1 5 1022011PRTHomo sapiens 220Ala Val Ser Asp Ala Gly Phe Gln Lys Leu Val1 5 1022112PRTHomo sapiens 221Ala Ala Thr Phe Thr Gly Gly Gly Asn Lys Leu Thr1 5 1022215PRTHomo sapiens 222Ala Val Ser Gly Met Asn Tyr Gly Gly Ser Gln Gly Asn Leu Ile1 5 10 1522310PRTHomo sapiens 223Ala Pro Tyr Thr Gly Arg Arg Ala Leu Thr1 5

1022410PRTHomo sapiens 224Val Val Asp Asn Tyr Gly Gln Asn Phe Val1 5 1022510PRTHomo sapiens 225Val Ala Asp Asn Tyr Gly Gln Asn Phe Val1 5 1022610PRTHomo sapiens 226Thr Ala Asp Asn Tyr Gly Gln Asn Phe Val1 5 102278PRTHomo sapiens 227Cys Gly Leu Trp Ser Glu Phe Cys1 52289PRTHomo sapiens 228Ala Trp Ile Thr Met Val Arg Ile Leu1 522910PRTHomo sapiens 229Ala Val Ser Asn Tyr Gly Gln Asn Phe Val1 5 102309PRTHomo sapiens 230Ala Val Ser Asn Asp Tyr Lys Leu Ser1 52319PRTHomo sapiens 231Ala Val Asp Ser Gly Gln Asn Phe Val1 52329PRTHomo sapiens 232Ala Val Asp Asn Tyr Val Arg Ile Leu1 523310PRTHomo sapiens 233Ala Val Asp Asn His Ser Gln Asn Phe Val1 5 1023411PRTHomo sapiens 234Ala Val Phe Gly Ser Asn Thr Gly Lys Leu Ile1 5 1023510PRTHomo sapiens 235Ala Leu Arg Ser Asn Asp Tyr Lys Leu Ser1 5 1023613PRTHomo sapiens 236Ala Val Arg Ile Ala Phe Trp Gly Leu Pro Glu Ser Tyr1 5 1023710PRTHomo sapiens 237Ala Ser Ser Leu Ala Gly Tyr Glu Gln Tyr1 5 1023811PRTHomo sapiens 238Ala Ser Ser Leu Gly Gln Gly Lys His Ser Phe1 5 1023913PRTHomo sapiens 239Ser Val Glu Gly Gly Ser Ser Gly Ala Asn Val Leu Thr1 5 1024010PRTHomo sapiens 240Ala Ser Ser Ser Ala Gly Tyr Glu Gln Tyr1 5 1024110PRTHomo sapiens 241Ala Ser Ser Leu Ala Gly Cys Glu Gln Tyr1 5 1024213PRTHomo sapiens 242Ala Ser Thr Ser Trp Gly Val Ser Tyr Asn Glu Gln Phe1 5 1024310PRTHomo sapiens 243Ala Ser Ser Leu Ala Ser Tyr Glu Gln Tyr1 5 1024410PRTHomo sapiens 244Ala Ser Ser Leu Ala Gly His Glu Gln Tyr1 5 1024513PRTHomo sapiens 245Ala Ser Ser Pro Pro Glu Gly Phe Gly Asn Glu Gln Phe1 5 1024610PRTHomo sapiens 246Ala Ser Ser Leu Thr Gly Tyr Glu Gln Tyr1 5 1024713PRTHomo sapiens 247Ser Ala Asn Leu Ala Lys Ser Ser Tyr Asn Glu Gln Phe1 5 1024812PRTHomo sapiens 248Ser Ala Pro Arg Asp Pro Asp Ala Asp Thr Gln Tyr1 5 1024911PRTHomo sapiens 249Ala Trp Asp Arg Thr Gly Glu Val Glu Gln Tyr1 5 102509PRTHomo sapiens 250Ala Ser Ser Ala Gly Tyr Glu Gln Tyr1 52519PRTHomo sapiens 251Ala Ala Ala Trp Pro Ala Thr Ser Ser1 525210PRTHomo sapiens 252Ala Ser Ser Met Ala Gly Tyr Glu Gln Tyr1 5 1025315PRTHomo sapiens 253Ala Trp Ser Phe His Pro Gly Leu Ala Ala Tyr Asn Glu Gln Phe1 5 10 1525412PRTHomo sapiens 254Ala Ser Ser Gln Leu Arg Gly Gly Ser Pro Leu His1 5 1025513PRTHomo sapiens 255Ala Ser Ser Gly Gln Gly Gly Ser Asn Thr Glu Ala Phe1 5 1025611PRTHomo sapiens 256Ala Ser Ser Leu Gly Gln Gly Arg His Ser Phe1 5 1025710PRTHomo sapiens 257Ala Ser Ser Leu Ala Glu Asp Thr Gln Tyr1 5 1025810PRTHomo sapiens 258Ala Ser Ser Trp Ala Gly Tyr Glu Gln Tyr1 5 1025911PRTHomo sapiens 259Ala Ser Ser Leu Gly Arg Gly Lys His Ser Phe1 5 1026010PRTHomo sapiens 260Ala Ser Ser Leu Ala Gly Phe Glu Gln Tyr1 5 102619PRTHomo sapiens 261Ala Ser Ser Leu Ala Gly Glu Gln Tyr1 526211PRTHomo sapiens 262Ala Ser Ser Leu Gly Gln Gly Lys Arg Ser Phe1 5 1026311PRTHomo sapiens 263Ala Ser Ser Leu Gly Gln Gly Glu His Ser Phe1 5 1026410PRTHomo sapiens 264Ala Tyr Ser Thr Gly Tyr Phe Gly Tyr Thr1 5 1026513PRTHomo sapiens 265Ser Val Gly Gly Gly Ser Ser Gly Ala Asn Val Leu Thr1 5 1026610PRTHomo sapiens 266Ala Ser Ser Val Ala Gly Tyr Glu Gln Tyr1 5 1026713PRTHomo sapiens 267Ser Val Glu Gly Gly Ser Pro Gly Ala Asn Val Leu Thr1 5 1026811PRTHomo sapiens 268Ala Ser Ser Ser Gly Gln Gly Lys His Ser Phe1 5 1026915PRTHomo sapiens 269Ala Ser Ser Glu Ser Arg Tyr Gly Arg Asp Thr Asp Thr Gln Tyr1 5 10 1527017PRTHomo sapiens 270Ala Ser Ser Tyr Ser Tyr Ser Thr Gly Pro Glu Leu Asn Thr Glu Ala1 5 10 15Phe27113PRTHomo sapiens 271Ser Val Glu Gly Gly Pro Ser Gly Ala Asn Val Leu Thr1 5 1027210PRTHomo sapiens 272Ala Ser Ser Leu Ala Gly Asn Glu Gln Tyr1 5 1027310PRTHomo sapiens 273Ala Ser Ser Leu Ala Ala Tyr Glu Gln Tyr1 5 1027411PRTHomo sapiens 274Ala Ser Ser Leu Ser Phe Asp Ser Glu Gln Tyr1 5 1027511PRTHomo sapiens 275Ala Ser Ser Leu Ser Gln Gly Lys His Ser Phe1 5 1027610PRTHomo sapiens 276Ala Ser Ser Phe Ala Gly Tyr Glu Gln Tyr1 5 1027710PRTHomo sapiens 277Ser Ala Arg Gln Gly Leu Thr Glu Ala Phe1 5 1027810PRTHomo sapiens 278Ala Trp Ser Val Leu Tyr Gly Thr Glu Tyr1 5 1027910PRTHomo sapiens 279Ala Ser Ser Leu Ala Gly Ser Glu Gln Tyr1 5 1028011PRTHomo sapiens 280Ser Val Glu Gly Asp Pro Leu Gly Pro Thr Ser1 5 1028110PRTHomo sapiens 281Ala Ser Ser Leu Ser Gly Tyr Glu Gln Tyr1 5 1028213PRTHomo sapiens 282Ser Val Glu Glu Gly Ser Ser Gly Ala Asn Val Leu Thr1 5 1028310PRTHomo sapiens 283Ala Ser Ser Leu Pro Gly Tyr Glu Gln Tyr1 5 1028413PRTHomo sapiens 284Ala Thr Ser Arg Glu Gly Thr Gly Glu Asn Ile Gln Tyr1 5 1028513PRTHomo sapiens 285Ala Ser Ser Phe Ser Ile Arg Ala Ser Tyr Glu Gln Tyr1 5 1028610PRTHomo sapiens 286Ala Ser Ser Leu Ala Gly Asp Glu Gln Tyr1 5 1028711PRTHomo sapiens 287Ala Ser Ser Val Ala Ser Thr Gly Glu Leu Phe1 5 1028812PRTHomo sapiens 288Ala Ser Ser Leu Gly Arg Gly Asn Thr Glu Ala Phe1 5 1028912PRTHomo sapiens 289Ala Ser Ser Pro Ile Arg Arg Glu Gly Glu Gln Tyr1 5 1029015PRTHomo sapiens 290Ala Ser Phe Val Tyr Ser Ala Gly Asp Ser Tyr Asn Glu Gln Phe1 5 10 1529110PRTHomo sapiens 291Ser Ala Arg Asn Arg Val Tyr Glu Gln Tyr1 5 1029212PRTHomo sapiens 292Ala Ser Ser Leu Gly Gln Gly Asn Thr Glu Ala Phe1 5 1029311PRTHomo sapiens 293Ala Ser Ser Pro Pro Gly Glu Asn Glu Gln Tyr1 5 1029411PRTHomo sapiens 294Ala Ser Thr Asp Thr Asp Leu Gly Glu Gln Tyr1 5 1029513PRTHomo sapiens 295Ser Val Glu Arg Gly Ser Ser Gly Ala Asn Val Leu Thr1 5 1029611PRTHomo sapiens 296Ala Ser Ser Leu Gly Gln Gly Asn His Ser Phe1 5 1029712PRTHomo sapiens 297Ala Ser Ser Leu Ala Arg Gly Asn Thr Glu Ala Phe1 5 1029812PRTHomo sapiens 298Ala Ala Ala Trp Ala Arg Gly Asn Thr Glu Ala Phe1 5 1029912PRTHomo sapiens 299Ala Leu Ser Asp Ser Gly Thr Ile Tyr Glu Gln Tyr1 5 1030017PRTHomo sapiens 300Ala Ser Ser Val Pro Leu Glu Gly Gly Ser Gly Pro Gln Asp Thr Gln1 5 10 15Tyr30111PRTHomo sapiens 301Ala Ser Ser Leu Gly Gln Gly Lys Tyr Ser Phe1 5 1030212PRTHomo sapiens 302Ala Ser Gly Leu Tyr Asn Arg Gly Asn Glu Gln Phe1 5 1030311PRTHomo sapiens 303Ser Val Glu Gly Gly Ser Ser Gly Pro Thr Ser1 5 1030410PRTHomo sapiens 304Ala Ser Gly Leu Ala Gly Tyr Glu Gln Tyr1 5 1030512PRTHomo sapiens 305Ala Ser Ser Arg Thr Arg Tyr Thr Asp Thr Gln Tyr1 5 1030610PRTHomo sapiens 306Ala Ser Asn Leu Ala Gly Tyr Glu Gln Tyr1 5 1030713PRTHomo sapiens 307Ala Ser Ala Ser Trp Gly Val Ser Tyr Asn Glu Gln Phe1 5 1030812PRTHomo sapiens 308Ala Ser Ser Leu Gly Gln Gly Glu Thr Leu Lys Leu1 5 1030913PRTHomo sapiens 309Ser Val Val Gly Gly Ser Ser Gly Ala Asn Val Leu Thr1 5 1031014PRTHomo sapiens 310Ser Val Gly Ala Asn Val Ala Gly Gly Lys Glu Thr Gln Tyr1 5 1031113PRTHomo sapiens 311Ala Ser Ser Val Thr Gly Thr Val Asn Thr Glu Ala Phe1 5 1031213PRTHomo sapiens 312Ser Val Lys Gly Gly Ser Ser Gly Ala Asn Val Leu Thr1 5 1031313PRTHomo sapiens 313Ser Val Glu Gly Gly Ser Thr Gly Ala Asn Val Leu Thr1 5 1031411PRTHomo sapiens 314Ala Ser Ser Leu Gly Gln Gly Ile His Ser Phe1 5 1031511PRTHomo sapiens 315Ala Ser Ser Leu Gly Gln Gly Lys Leu Ser Phe1 5 1031611PRTHomo sapiens 316Ala Ser Ser Gln Asp Ile Glu Val Glu Ala Phe1 5 1031711PRTHomo sapiens 317Ala Ser Ser Leu Arg Leu Asn Thr Glu Ala Phe1 5 1031814PRTHomo sapiens 318Ala Ser Ser Val Glu Ala Gly Val Ser Gly Asn Thr Ile Tyr1 5 1031911PRTHomo sapiens 319Ser Val Val Arg Gln Gly His Tyr Glu Ala Phe1 5 1032013PRTHomo sapiens 320Ser Val Glu Gly Gly Ser Phe Gly Ala Asn Val Leu Thr1 5 1032113PRTHomo sapiens 321Ala Ser Ser Pro Gly Arg Ile Leu Thr Asp Thr Gln Tyr1 5 1032210PRTHomo sapiens 322Ala Ser Ser Leu Ala Asp Tyr Glu Gln Tyr1 5 1032311PRTHomo sapiens 323Ala Ser Ser Gly Gly Leu Asn Gln Pro Gln His1 5 1032412PRTHomo sapiens 324Ala Ser Lys Val Gln Gly Ser Glu Asp Thr Gln Tyr1 5 1032513PRTHomo sapiens 325Ala Ser Ser Pro Pro Gly Gly Phe Gly Asn Glu Gln Phe1 5 1032614PRTHomo sapiens 326Ala Ser Ser Gly Ala Gly Gln Gly Ser Ser Tyr Glu Gln Tyr1 5 1032710PRTHomo sapiens 327Ala Ser Ser Leu Ala Ser Cys Glu Gln Tyr1 5 1032810PRTHomo sapiens 328Ala Ser Ser Leu Ala Gly Tyr Arg Gln Tyr1 5 1032910PRTHomo sapiens 329Ala Ser Ser Leu Ala Gly Tyr Lys Gln Tyr1 5 1033012PRTHomo sapiens 330Ala Ser Thr Ser Trp Ala Ser Pro Thr Met Ser Ser1 5 1033113PRTHomo sapiens 331Ala Ser Thr Ser Arg Gly Val Ser Tyr Asn Glu Gln Phe1 5 1033211PRTHomo sapiens 332Ala Ser Ser Phe Gly Gln Gly Lys His Ser Phe1 5 1033310PRTHomo sapiens 333Ala Ser Ser Gly Gln Gly Lys His Ser Phe1 5 1033410PRTHomo sapiens 334Ala Ser Ser Leu Val Gly His Glu Gln Tyr1 5 1033510PRTHomo sapiens 335Ala Ser Ser Leu Ala Gly His Gly Gln Tyr1 5 1033610PRTHomo sapiens 336Ala Ser Asn Ser Ala Gly Tyr Glu Gln Tyr1 5 1033710PRTHomo sapiens 337Ala Ser Gly Ser Ala Gly Tyr Glu Gln Tyr1 5 1033813PRTHomo sapiens 338Ala Ser Thr Ser Trp Gly Ile Ser Tyr Asn Glu Gln Phe1 5 1033913PRTHomo sapiens 339Ala Ser Thr Ser Trp Gly Ala Ser Tyr Asn Glu Gln Phe1 5 1034012PRTHomo sapiens 340Ala Ser Thr Ser Gly Val Ser Tyr Asn Glu Gln Phe1 5 1034113PRTHomo sapiens 341Ala Gly Thr Ser Trp Gly Val Ser Tyr Asn Glu Gln Phe1 5 1034211PRTHomo sapiens 342Ala Ser Ser Val Gly Gln Gly Lys His Ser Phe1 5 1034311PRTHomo sapiens 343Ala Ser Ser Met Gly Gln Gly Lys His Ser Phe1 5 1034411PRTHomo sapiens 344Ala Ser Ser Leu Cys Gln Gly Lys His Ser Phe1 5 1034512PRTHomo sapiens 345Ala Ser Arg Gly Leu Ala Gly Phe Asn Glu Gln Phe1 5 1034615PRTHomo sapiens 346Ala Ser Ser Leu Met Arg Val Gly Phe Arg Thr Asp Thr Gln Tyr1 5 10 1534713PRTHomo sapiens 347Ala Ser Ser Gln Asp Glu Leu Ala Gly Arg Thr Gln Tyr1 5 1034813PRTHomo sapiens 348Ala Ser Thr Asn Ser Leu Thr Ser Thr Asp Thr Gln Tyr1 5 1034913PRTHomo sapiens 349Ala Ser Ser Pro Pro Glu Gly Leu Gly Asn Glu Gln Phe1 5 1035011PRTHomo sapiens 350Ala Ile Ser Arg Ala Asp Gln Glu Thr Gln Tyr1 5 1035112PRTHomo sapiens 351Ser Ala Arg Asp Arg Gly Ala Thr Gly Glu Leu Phe1 5 1035213PRTHomo sapiens 352Ser Val Glu Gly Gly Ser Ala Gly Ala Asn Val Leu Thr1 5 1035310PRTHomo sapiens 353Ser Ala Arg Gln Gly Arg Thr Glu Ala Phe1 5 1035413PRTHomo sapiens 354Ala Ser Thr Ser Trp Gly Val Pro Tyr Asn Glu Gln Phe1 5 1035510PRTHomo sapiens 355Ala Ser Ser Leu Thr Ser Tyr Glu Gln Tyr1 5 1035610PRTHomo sapiens 356Ala Ser Ser Leu Thr Gly Cys Glu Gln Tyr1 5 1035711PRTHomo sapiens 357Ala Ser Ser Leu Gly Gln Arg Lys His Ser Phe1 5 103589PRTHomo sapiens 358Ala Ser Ser Leu Ala Gly Tyr Lys Tyr1 535910PRTHomo sapiens 359Ala Ser Gly Met Ala Gly Tyr Glu Gln Tyr1 5 1036012PRTHomo sapiens 360Ala Ser Ser Trp Ala Arg Gly Asn Thr Glu Ala Phe1 5 1036111PRTHomo sapiens 361Ala Ser Ser Leu Gly Gln Glu Thr Leu Lys Leu1 5 103629PRTHomo sapiens 362Ala Ser Thr Leu Tyr Glu Lys Leu Phe1 536310PRTHomo sapiens 363Ala Ser Asp Leu Ala Gly Tyr Glu Gln Tyr1 5 1036412PRTHomo sapiens 364Ala Ser Val Gly Thr Gly Asn Val Asp Glu Gln Tyr1 5 1036513PRTHomo sapiens 365Ala Ser Ser Pro Pro Glu Gly Ser Gly Asn Glu Gln Phe1 5 1036613PRTHomo sapiens 366Ala Ser Ser Pro Pro Glu Gly Phe Ser Asn Glu Gln Phe1 5 1036713PRTHomo sapiens 367Ser Ala Asn Leu Ala Arg Ser Ser Tyr Asn Glu Gln Phe1 5 1036813PRTHomo sapiens 368Ser Ala Leu Asp Leu Ala Gly Ser Gln Glu Thr Gln Tyr1 5 1036912PRTHomo sapiens 369Ala Val Glu Leu Phe Ala Ala Gly Asn Lys Leu Thr1 5 1037012PRTHomo sapiens 370Ala Gly Ala Val Thr Gly Gln Leu Gln Gln Ile Leu1 5 1037112PRTHoJo virus 371Ala Val Gly Leu Phe Ala Ala Gly Asn Lys Leu Thr1 5 1037212PRTHomo sapiens 372Ala Val Glu Leu Leu Ala Ala Gly Asn Lys Leu Thr1 5 1037312PRTHomo sapiens 373Ala Val Glu Leu Ser Ala Ala Gly Asn Lys Leu Thr1 5 1037412PRTHomo sapiens 374Ala Val Lys Leu Phe Ala Ala Gly Asn Lys Leu Thr1 5 1037512PRTHomo sapiens 375Ala Val Glu Leu Phe Thr Ala Gly Asn Lys Leu Thr1 5 1037612PRTHomo sapiens 376Ala Val Glu Leu Phe Ala Thr Gly Asn Lys Leu Thr1 5 1037712PRTHomo sapiens 377Ala Val Glu Leu Phe Val Ala Gly Asn Lys Leu Thr1 5 1037811PRTHomo sapiens 378Ala Val Glu Leu Phe Ala Ala Gly Asn Lys Leu1 5 1037912PRTHomo sapiens 379Ala Val Val Leu Phe Ala Ala Gly Asn Lys Leu Thr1 5 1038010PRTHomo sapiens 380Ala Val Ser Tyr Leu Leu Gln Ala Thr Ser1 5 1038111PRTHomo sapiens 381Ala Val Leu Phe Ala Ala Gly Asn Lys Leu Thr1 5 1038212PRTHomo sapiens 382Ala Val Glu Leu Phe Ala Ser Gly Asn Lys Leu Thr1 5 1038310PRTHomo sapiens 383Ala Val Glu Leu Phe Ala Ala Ala Thr Ser1 5 1038412PRTHomo sapiens 384Ala Val Asp Leu Phe Ala Ala Gly Asn Lys Leu Thr1 5 1038512PRTHomo sapiens 385Ala Val Glu Leu Phe Asp Ala Gly Asn Lys Leu Thr1 5 1038611PRTHomo sapiens 386Ala Ala Ser Glu Gly Asn Tyr Asn Val Leu Tyr1 5 1038712PRTHomo sapiens 387Ala Val Glu Val Phe Ala Ala Gly Asn Lys Leu Thr1 5 1038812PRTHomo sapiens 388Ala Val Glu Leu Phe Ser Ala Gly Asn Lys Leu Thr1 5 1038912PRTHomo sapiens 389Ala Val Ala Leu Phe Ala Ala Gly Asn Lys Leu Thr1 5 1039012PRTHomo sapiens 390Ala Val Glu Ile Phe Ala Ala Gly Asn Lys Leu Thr1 5 1039112PRTHomo sapiens 391Ala Val Glu Leu Phe Pro Ala Gly Asn Lys Leu Thr1 5 1039212PRTHomo sapiens 392Ala Val Glu Leu Phe Gly Ala Gly Asn Lys Leu Thr1 5 1039310PRTHomo sapiens 393Ala Val Glu Leu Phe Ala Glu Ala Thr Ser1 5 1039413PRTHomo sapiens 394Ala Ser Ser Leu Ile Ser Gly Ser Ser Tyr Glu Gln Tyr1 5 1039512PRTHomo sapiens 395Ala Ser

Ser Ile Glu Gly Gln Lys Thr Leu Lys Leu1 5 1039613PRTHomo sapiens 396Ala Ser Ser Leu Ile Ser Gly Ser Pro Tyr Glu Gln Tyr1 5 1039713PRTHomo sapiens 397Ala Ser Gly Leu Ile Ser Gly Ser Ser Tyr Glu Gln Tyr1 5 1039813PRTHomo sapiens 398Ala Ser Ser Leu Ile Gly Gly Ser Ser Tyr Glu Gln Tyr1 5 1039913PRTHomo sapiens 399Ala Ser Ser Leu Val Ser Gly Ser Ser Tyr Glu Gln Tyr1 5 1040013PRTHomo sapiens 400Ala Ser Ser Pro Ile Ser Gly Ser Ser Tyr Glu Gln Tyr1 5 1040113PRTHomo sapiens 401Ala Ser Ser Leu Ile Ser Gly Gly Ser Tyr Glu Gln Tyr1 5 1040213PRTHomo sapiens 402Ala Ser Asn Leu Ile Ser Gly Ser Ser Tyr Glu Gln Tyr1 5 1040313PRTHomo sapiens 403Ala Ser Ser Leu Ile Ser Gly Ser Phe Tyr Glu Gln Tyr1 5 1040413PRTHomo sapiens 404Ala Ser Ser Leu Ile Ser Gly Ser Thr Tyr Glu Gln Tyr1 5 1040512PRTHomo sapiens 405Ala Ser Ser Ile Glu Gly Gln Lys Ala Leu Lys Leu1 5 1040613PRTHomo sapiens 406Ala Ser Arg Leu Ile Ser Gly Ser Ser Tyr Glu Gln Tyr1 5 1040713PRTHomo sapiens 407Ala Ser Ser Ile Glu Gly Gln Lys Trp Thr Leu Lys Leu1 5 1040810PRTHomo sapiens 408Ala Ala Val Val Val Ala Pro Thr Ser Ser1 5 1040912PRTHomo sapiens 409Ala Ser Ser Ile Gly Gly Gln Lys Thr Leu Lys Leu1 5 1041012PRTHomo sapiens 410Ala Ser Ser Ile Glu Gly Gln Arg Thr Leu Lys Leu1 5 1041112PRTHomo sapiens 411Ala Ser Ser Ile Glu Gly Arg Lys Thr Leu Lys Leu1 5 1041212PRTHomo sapiens 412Ala Ser Ser Thr Glu Gly Gln Lys Thr Leu Lys Leu1 5 1041312PRTHomo sapiens 413Ala Ser Ser Leu Ile Ser Val Ala Pro Thr Ser Ser1 5 1041412PRTHomo sapiens 414Ala Ser Ser Ile Glu Gly Gln Glu Thr Leu Lys Leu1 5 1041513PRTHomo sapiens 415Ala Arg Ser Leu Ile Ser Gly Ser Ser Tyr Glu Gln Tyr1 5 1041612PRTHomo sapiens 416Ala Ser Ser Met Glu Gly Gln Lys Thr Leu Lys Leu1 5 1041712PRTHomo sapiens 417Ala Ser Ser Ile Glu Glu Gln Lys Thr Leu Lys Leu1 5 1041813PRTHomo sapiens 418Ala Ser Ser Ile Glu Gly Gln Lys Arg Thr Leu Lys Leu1 5 1041913PRTHomo sapiens 419Ala Ser Ser Leu Ile Ser Gly Ser Ala Tyr Glu Gln Tyr1 5 1042013PRTHomo sapiens 420Ala Ser Cys Leu Ile Ser Gly Ser Ser Tyr Glu Gln Tyr1 5 1042113PRTHomo sapiens 421Ala Ser Ser Leu Ile Ser Gly Arg Ser Tyr Glu Gln Tyr1 5 1042213PRTHomo sapiens 422Ala Ser Ser Leu Ile Ser Gly Ser Tyr Tyr Glu Gln Tyr1 5 1042313PRTHomo sapiens 423Ala Ser Ser Ile Glu Gly Gln Lys Cys Thr Leu Lys Leu1 5 1042412PRTHomo sapiens 424Ala Ser Ser Leu Ser Gly Ser Ser Tyr Glu Gln Tyr1 5 1042512PRTHomo sapiens 425Ala Ser Ser Leu Ile Ser Gly Ser Ser Thr Ser Ser1 5 1042613PRTHomo sapiens 426Ala Ser Ser Leu Ile Ser Gly Ser Ser Arg Glu Gln Tyr1 5 1042713PRTHomo sapiens 427Ala Ser Ile Leu Ile Ser Gly Ser Ser Tyr Glu Gln Tyr1 5 1042812PRTHomo sapiens 428Ala Ser Ser Lys Glu Gly Gln Lys Thr Leu Lys Leu1 5 1042913PRTHomo sapiens 429Ala Ser Thr Leu Ile Ser Gly Ser Ser Tyr Glu Gln Tyr1 5 1043013PRTHomo sapiens 430Ala Ser Ser Pro Ile Ser Gly Ser Pro Tyr Glu Gln Tyr1 5 1043113PRTHomo sapiens 431Ala Ser Ser Leu Val Ser Gly Asn Ser Tyr Glu Gln Tyr1 5 1043213PRTHomo sapiens 432Ala Ser Ser Leu Ile Ser Gly Ser Cys Tyr Glu Gln Tyr1 5 1043313PRTHomo sapiens 433Ala Ser Gly Pro Ile Ser Gly Ser Ser Tyr Glu Gln Tyr1 5 1043413PRTHomo sapiens 434Ala Ser Gly Leu Ile Ser Gly Gly Ser Tyr Glu Gln Tyr1 5 1043513PRTHomo sapiens 435Ala Ser Gly Leu Ile Gly Gly Ser Ser Tyr Glu Gln Tyr1 5 1043611PRTHomo sapiens 436Ala Ser Ser Ile Glu Gly Gln Lys His Ser Phe1 5 1043712PRTHomo sapiens 437Ala Ser Ser Arg Glu Gly Gln Lys Thr Leu Lys Leu1 5 1043812PRTHomo sapiens 438Ala Ser Ser Ile Lys Gly Gln Lys Thr Leu Lys Leu1 5 1043913PRTHomo sapiens 439Ala Ser Ser Ile Glu Gly Gln Lys Gly Thr Leu Lys Leu1 5 1044011PRTHomo sapiens 440Ala Pro Pro Pro Gly Ser Ala Arg Gln Leu Thr1 5 104419PRTHomo sapiens 441Ala Val Met Asn Ala Gly Lys Ser Thr1 544211PRTHomo sapiens 442Ala Leu Pro Ser Gly Ser Ala Arg Gln Leu Thr1 5 1044311PRTHomo sapiens 443Ala Pro Pro Ser Ser Ser Ala Arg Gln Leu Thr1 5 1044411PRTHomo sapiens 444Thr Pro Pro Ser Gly Ser Ala Arg Gln Leu Thr1 5 1044511PRTHomo sapiens 445Val Pro Pro Ser Gly Ser Ala Arg Gln Leu Thr1 5 1044611PRTHomo sapiens 446Ala Pro Ser Ser Gly Ser Ala Arg Gln Leu Thr1 5 1044711PRTHomo sapiens 447Ala Pro Pro Phe Gly Ser Ala Arg Gln Leu Thr1 5 1044811PRTHomo sapiens 448Ala Pro Pro Thr Gly Ser Ala Arg Gln Leu Thr1 5 1044911PRTHomo sapiens 449Ala Glu Asn Glu Asp Tyr Gly Gln Asn Phe Val1 5 1045010PRTHomo sapiens 450Ala Pro Pro Ser Gly Ser Ala Arg Gln Leu1 5 1045152PRTHomo sapiens 451Ala Pro Pro Ser Gly Ser Ala Arg Gln Leu Thr Phe Gly Ser Gly Thr1 5 10 15Gln Leu Thr Val Leu Pro Glu His Ile Lys Lys Arg Gly Glu Val Thr 20 25 30Lys Gly Ser Leu Leu Gly Ile Lys His Cys Asp Thr His Gly Arg Arg 35 40 45Lys Gln Thr His 5045211PRTHomo sapiens 452Ala Gln Pro Ser Gly Ser Ala Arg Gln Leu Thr1 5 1045311PRTHomo sapiens 453Ala Pro Pro Ser Cys Ser Ala Arg Gln Leu Thr1 5 1045411PRTHomo sapiens 454Ala Pro Pro Ala Gly Ser Ala Arg Gln Leu Thr1 5 1045511PRTHomo sapiens 455Ala Pro Pro Tyr Gly Ser Ala Arg Gln Leu Thr1 5 1045611PRTHomo sapiens 456Asp Pro Pro Ser Gly Ser Ala Arg Gln Leu Thr1 5 1045711PRTHomo sapiens 457Gly Pro Pro Ser Gly Ser Ala Arg Gln Leu Thr1 5 1045812PRTHomo sapiens 458Ala Val Asn Ile Gly Gly Ser Gln Gly Asn Leu Ile1 5 1045911PRTHomo sapiens 459Ser Pro Pro Ser Gly Ser Ala Arg Gln Leu Thr1 5 1046011PRTHomo sapiens 460Ala Pro Pro Ser Arg Ser Ala Arg Gln Leu Thr1 5 104619PRTHomo sapiens 461Ala Arg Pro Leu Val Leu Gln Gly Asn1 546211PRTHomo sapiens 462Pro Pro Pro Ser Gly Ser Ala Arg Gln Leu Thr1 5 1046311PRTHomo sapiens 463Ala Pro Pro Ser Asp Ser Ala Arg Gln Leu Thr1 5 1046410PRTHomo sapiens 464Ala Val Tyr Ser Gly Tyr Ser Thr Leu Thr1 5 1046513PRTHomo sapiens 465Ala Val Asn Ala Arg Asp Ser Gly Thr Tyr Lys Tyr Ile1 5 1046612PRTHomo sapiens 466Ala Ala Pro Gly Glu Cys Trp Gln Gln Pro Ala Asp1 5 1046714PRTHomo sapiens 467Ala Arg Thr Gly Tyr Ser Gly Gly Gly Ala Asp Gly Leu Thr1 5 1046811PRTHomo sapiens 468Ala Tyr Asp Gln Gly Gly Ser Glu Lys Leu Val1 5 1046912PRTHomo sapiens 469Ala Met Ser Phe Arg Gly Gly Tyr Gln Lys Val Thr1 5 1047011PRTHomo sapiens 470Ala Pro Pro Ser Gly Pro Ala Arg Gln Leu Thr1 5 1047111PRTHomo sapiens 471Ala Pro Pro Ser Asp Pro Ala Arg Gln Leu Thr1 5 1047211PRTHomo sapiens 472Ala Pro Pro Cys Gly Ser Ala Arg Gln Leu Thr1 5 1047311PRTHomo sapiens 473Ala Val Arg Pro Ile Glu His Arg Pro Val Leu1 5 1047411PRTHomo sapiens 474Ala Leu Arg Ser Gly Gly Tyr Gln Lys Val Thr1 5 104759PRTHomo sapiens 475Ala Val Gly Ala Gly Gly Lys Leu Ile1 54769PRTHomo sapiens 476Ala Val Thr Asn Ala Gly Lys Ser Thr1 54779PRTHomo sapiens 477Ala Val Met Ser Ala Gly Lys Ser Thr1 54789PRTHomo sapiens 478Ala Val Met Asp Ala Gly Lys Ser Thr1 54797PRTHomo sapiens 479Pro Leu Val Leu Gln Gly Asn1 548052PRTHomo sapiens 480Ala Pro Pro Ser Gly Ser Ala Arg Gln Leu Thr Phe Gly Ser Gly Thr1 5 10 15Gln Leu Thr Val Leu Pro Glu His Ile Lys Lys Arg Gly Glu Val Thr 20 25 30Lys Gly Ser Leu Leu Gly Ile Lys His Cys Glu Thr His Gly Arg Arg 35 40 45Lys Gln Thr His 5048111PRTHomo sapiens 481Ala Pro Pro Pro Asp Ser Ala Arg Gln Leu Thr1 5 1048212PRTHomo sapiens 482Ala Met Ser Leu Asp Asn Tyr Gly Gln Asn Phe Val1 5 1048311PRTHomo sapiens 483Ala Phe Ile Leu Gln Gly Ala Gln Lys Leu Val1 5 1048410PRTHomo sapiens 484Ala Leu Glu Leu Ser Gly Tyr Ala Leu Asn1 5 1048510PRTHomo sapiens 485Ile Trp Leu Arg Ala Asp Leu Lys Ser Trp1 5 1048611PRTHomo sapiens 486Ala Tyr Gly Ser Ser Asn Thr Gly Lys Leu Ile1 5 1048710PRTHomo sapiens 487Ala Val Ser Ala Arg Arg Gln Asn Phe Val1 5 104889PRTHomo sapiens 488Ala Val Arg Asn Ala Gly Lys Ser Thr1 54899PRTHomo sapiens 489Ala Met Met Asn Ala Gly Lys Ser Thr1 549011PRTHomo sapiens 490Val Pro Ser Ser Gly Ser Ala Arg Gln Leu Thr1 5 1049111PRTHomo sapiens 491Thr Pro Pro Phe Gly Ser Ala Arg Gln Leu Thr1 5 1049211PRTHomo sapiens 492Thr Pro Leu Ser Gly Ser Ala Arg Gln Leu Thr1 5 1049310PRTHomo sapiens 493Ser Ala Leu Trp Phe Cys Lys Ala Thr Asp1 5 1049411PRTHomo sapiens 494Ala Thr Pro Pro Gly Ser Ala Arg Gln Leu Thr1 5 1049511PRTHomo sapiens 495Ala Ser Pro Pro Gly Ser Ala Arg Gln Leu Thr1 5 1049612PRTHomo sapiens 496Ala Pro Ser Ser Ala Gly Asn Asn Arg Lys Leu Ile1 5 104979PRTHomo sapiens 497Ala Pro Pro Ser Val Leu Gln Gly Asn1 549852PRTHomo sapiens 498Ala Pro Pro Ser Gly Ser Ala Arg Gln Leu Thr Phe Gly Ser Gly Thr1 5 10 15Gln Leu Ala Val Leu Pro Glu His Ile Lys Lys Arg Gly Glu Val Thr 20 25 30Lys Gly Ser Leu Leu Gly Ile Lys His Cys Asp Thr His Gly Arg Arg 35 40 45Lys Gln Thr His 5049910PRTHomo sapiens 499Ala Pro Pro Ser Gly Ser Ala Gly Asn Trp1 5 1050011PRTHomo sapiens 500Ala Pro Pro Ser Gly Leu Ala Arg Gln Leu Thr1 5 1050110PRTHomo sapiens 501Ala Pro Pro Pro Gly Ser Ala Arg Gln Leu1 5 1050211PRTHomo sapiens 502Ala Pro Pro Leu Gly Ser Ala Arg Gln Leu Thr1 5 1050311PRTHomo sapiens 503Ala Leu Pro Pro Gly Ser Ala Arg Gln Leu Thr1 5 1050410PRTHomo sapiens 504Ala Leu Asp Leu Thr Gly Asn Gln Phe Tyr1 5 1050513PRTHomo sapiens 505Ala Ala Ser Leu Ser Asn Phe Gly Asn Glu Lys Leu Thr1 5 1050614PRTHomo sapiens 506Ala Ala Ser Phe Ser Asp Gln Thr Gly Ala Asn Asn Leu Phe1 5 1050713PRTHomo sapiens 507Ala Ser Ser Pro Pro Glu Gly Phe Gly Asp Glu Gln Phe1 5 1050813PRTHomo sapiens 508Ala Ser Ser Pro Pro Glu Gly Phe Gly Ser Glu Gln Phe1 5 1050913PRTHomo sapiens 509Ala Ser Ser Pro Pro Glu Gly Phe Asp Asn Glu Gln Phe1 5 1051013PRTHomo sapiens 510Ala Ser Ser Leu Pro Glu Gly Phe Gly Asn Glu Gln Phe1 5 1051113PRTHomo sapiens 511Ala Ser Ser Pro Leu Glu Gly Phe Gly Asn Glu Gln Phe1 5 1051213PRTHomo sapiens 512Ala Ser Ser Pro Ser Glu Gly Phe Gly Asn Glu Gln Phe1 5 1051312PRTHomo sapiens 513Ala Ser Ser Pro Pro Glu Gly Leu Ala Met Ser Ser1 5 1051412PRTHomo sapiens 514Ala Ser Ser His Leu Arg Val Leu Ala Met Ser Ser1 5 1051512PRTHomo sapiens 515Ala Ser Ser Pro Pro Gly Leu Gly Tyr Glu Gln Tyr1 5 1051612PRTHomo sapiens 516Ala Ser Ser Pro Arg Gly Gly Ser Tyr Glu Gln Tyr1 5 1051713PRTHomo sapiens 517Ala Ser Ser Pro Pro Glu Gly Phe Gly Lys Glu Gln Phe1 5 1051812PRTHomo sapiens 518Ala Ile Arg Ser Thr Ala Ser Thr Asp Thr Gln Tyr1 5 1051913PRTHomo sapiens 519Ala Ser Ser Pro His Glu Gly Phe Gly Asn Glu Gln Phe1 5 1052012PRTHomo sapiens 520Ala Ala His His Leu Arg Val Leu Ala Met Ser Ser1 5 1052113PRTHomo sapiens 521Ala Ser Ser Pro Pro Glu Gly Phe Gly Tyr Glu Gln Phe1 5 1052213PRTHomo sapiens 522Ala Ser Ser Gln Pro Glu Gly Phe Gly Asn Glu Gln Phe1 5 1052311PRTHomo sapiens 523Ala Ser Arg Gly Thr Ser Val Gln Gln Ala Val1 5 1052413PRTHomo sapiens 524Ala Ser Thr Pro Ser Gly Pro Ser Thr Asp Thr Gln Tyr1 5 1052513PRTHomo sapiens 525Ala Ser Ser Pro Pro Glu Gly Phe Gly Ile Glu Gln Phe1 5 1052615PRTHomo sapiens 526Ala Ser Ser Tyr Ser Gly Ala Gly Gly Pro Trp Asp Thr Gln Tyr1 5 10 1552713PRTHomo sapiens 527Ala Ser Ser Pro Pro Glu Gly Phe Gly Thr Glu Gln Phe1 5 1052813PRTHomo sapiens 528Ala Ser Ser Val Glu Gly Thr Gly Thr Ser Ile Gln Tyr1 5 1052913PRTHomo sapiens 529Ala Ser Ser Pro Pro Glu Gly Phe Gly His Glu Gln Phe1 5 1053013PRTHomo sapiens 530Ala Ser Ser Pro Thr Glu Gly Phe Gly Asn Glu Gln Phe1 5 1053110PRTHomo sapiens 531Ala Ser Ser Leu Trp Gly Thr Glu Ala Phe1 5 1053213PRTHomo sapiens 532Ala Ser Ser Arg Pro Glu Gly Phe Gly Asn Glu Gln Phe1 5 1053313PRTHomo sapiens 533Ala Ser Ser Pro Arg Glu Gly Phe Gly Asn Glu Gln Phe1 5 1053412PRTHomo sapiens 534Ala Ser Ser Leu Asp Arg Leu Tyr Thr Glu Ala Phe1 5 1053512PRTHomo sapiens 535Ala Ser Ser Phe Gly Thr Gly Gly Asn Thr Gln Tyr1 5 1053613PRTHomo sapiens 536Ala Ser Ser Pro Ala Glu Gly Phe Gly Asn Glu Gln Phe1 5 1053711PRTHomo sapiens 537Ala Ser Ser Leu Tyr Phe Gly Gln Pro Gln His1 5 1053810PRTHomo sapiens 538Ala Ser Arg Thr Glu Ala Arg Glu Gln Tyr1 5 1053911PRTHomo sapiens 539Ala Ser Ser Gln Pro Gly Gln Tyr Gly Tyr Thr1 5 1054013PRTHomo sapiens 540Ala Ser Ser Pro Pro Glu Gly Phe Gly Asp Gly Gln Phe1 5 1054110PRTHomo sapiens 541Ala Ser Arg Leu Gly His Gln Pro Gln His1 5 1054213PRTHomo sapiens 542Ala Ser Ser Pro Pro Lys Gly Phe Gly Asn Glu Gln Phe1 5 1054313PRTHomo sapiens 543Ala Ser Ser Pro Pro Glu Gly Phe Gly Asn Arg Gln Phe1 5 1054413PRTHomo sapiens 544Ala Ser Pro Pro Pro Glu Gly Phe Gly Asn Glu Gln Phe1 5 1054515PRTHomo sapiens 545Ser Val Gly Val Thr Gly Gly Thr Ile Thr Pro His Glu Gln Tyr1 5 10 1554617PRTHomo sapiens 546Ala Ser Ser Asp Arg Asp Arg Asp Gly Arg Ala Arg Gly Gly Glu Gln1 5 10 15Phe54713PRTHomo sapiens 547Ala Ser Ser Pro Pro Lys Gly Leu Gly Asn Glu Gln Phe1 5 1054813PRTHomo sapiens 548Ala Ser Ser Pro Pro Glu Ser Leu Gly Asn Glu Gln Phe1 5 1054913PRTHomo sapiens 549Ala Ser Ser Pro Pro Glu Gly Phe Gly Asn Lys Gln Phe1 5 1055012PRTHomo sapiens 550Ala Trp Arg Pro Gly Tyr Met Asn Thr Glu Ala Phe1 5 1055115PRTHomo sapiens 551Ala Ile Ser Glu Trp Ala Ser Gly Arg Pro Ser Tyr Glu Gln Tyr1 5 10 1555213PRTHomo sapiens 552Ala Ser Ser Ala Leu Ala Gly Asp Thr Tyr Glu Gln Tyr1 5 1055312PRTHomo sapiens 553Ala Ser Ser Pro Arg Arg Gly Ser Tyr Glu Gln Tyr1 5 1055411PRTHomo sapiens 554Ala Ser Ser Pro Arg Gly Ala Pro Thr Ser Ser1 5 1055513PRTHomo sapiens 555Ala Ser Ser Tyr Ser Pro Gly Asn His Gln Pro Gln His1 5

1055613PRTHomo sapiens 556Ala Ser Ser Pro Ser Glu Gly Phe Asp Asn Glu Gln Phe1 5 1055713PRTHomo sapiens 557Ala Ser Ser Pro Pro Glu Gly Leu Ser Asn Glu Gln Phe1 5 1055813PRTHomo sapiens 558Ala Ser Ser Pro Pro Glu Gly Phe Ser Ser Glu Gln Phe1 5 1055912PRTHomo sapiens 559Ala Ser Ser Pro Pro Glu Gly Phe Gly Asn Glu Phe1 5 1056013PRTHomo sapiens 560Ala Ser Ser Pro Pro Glu Asp Phe Gly Asn Glu Gln Phe1 5 1056112PRTHomo sapiens 561Ala Ile Arg Ser Thr Ala Ser Ala Asp Thr Gln Tyr1 5 1056212PRTHomo sapiens 562Ala Thr Thr Arg Thr Ser Gly Ser Asn Glu Gln Phe1 5 1056312PRTHomo sapiens 563Ala Ser Ser Pro Arg Gly Gly Leu Leu Arg Ala Val1 5 1056413PRTHomo sapiens 564Ala Ser Ser Tyr Gly Leu Ala His Ser Tyr Glu Gln Tyr1 5 1056510PRTHomo sapiens 565Ala Thr Leu Gln Gly Pro Asn Glu Gln Phe1 5 1056614PRTHomo sapiens 566Ala Ser Ser Ser Ser Val Leu Arg Ala Arg Thr Glu Ala Phe1 5 1056714PRTHomo sapiens 567Ala Ser Ser Ile Phe Leu Gly Asp Asn Thr Gly Glu Leu Phe1 5 1056813PRTHomo sapiens 568Ala Ser Leu Pro Pro Glu Gly Phe Gly Asn Glu Gln Phe1 5 1056913PRTHomo sapiens 569Ala Ser Ser Pro Ser Glu Asp Phe Gly Asn Glu Gln Phe1 5 1057013PRTHomo sapiens 570Ala Ser Ser Pro Pro Lys Gly Ser Gly Asn Glu Gln Phe1 5 1057113PRTHomo sapiens 571Ala Ser Ser Pro Pro Gly Gly Phe Asp Asn Glu Gln Phe1 5 1057213PRTHomo sapiens 572Ala Ser Ser Pro Pro Glu Gly Phe Asn Asn Glu Gln Phe1 5 1057313PRTHomo sapiens 573Ala Ser Ser Pro Pro Glu Gly Phe Gly Tyr Gly Gln Phe1 5 1057413PRTHomo sapiens 574Ala Ser Ser Pro Pro Glu Gly Phe Gly Asn Gly Gln Phe1 5 1057513PRTHomo sapiens 575Ala Ser Ser Pro Pro Glu Asp Ser Gly Asn Glu Gln Phe1 5 1057613PRTHomo sapiens 576Ala Ser Ser Pro Pro Glu Asp Phe Asp Asn Glu Gln Phe1 5 1057713PRTHomo sapiens 577Ala Ser Ser Pro Leu Lys Gly Phe Gly Asn Glu Gln Phe1 5 1057813PRTHomo sapiens 578Ala Ser Ser Pro Leu Gly Gly Phe Gly Asn Glu Gln Phe1 5 1057913PRTHomo sapiens 579Ala Ser Ser Pro Leu Glu Gly Phe Ser Asn Glu Gln Phe1 5 1058013PRTHomo sapiens 580Ala Ser Ser Pro Leu Glu Asp Phe Gly Asn Glu Gln Phe1 5 1058113PRTHomo sapiens 581Ala Ser Ser Leu Pro Glu Gly Phe Asp Asn Glu Gln Phe1 5 1058213PRTHomo sapiens 582Ala Ser Pro Leu Pro Glu Gly Phe Gly Asn Glu Gln Phe1 5 1058310PRTHomo sapiens 583Ser Val Trp Thr Asp Ser Asp Thr Gln Tyr1 5 1058411PRTHomo sapiens 584Ala Ser Ser Gly Pro Ser Gly Gln Pro Gln His1 5 1058517PRTHomo sapiens 585Ala Ser Leu Ser Ser Pro Pro Arg Asp Pro Trp Arg Leu Ile His Pro1 5 10 15Ser58611PRTHomo sapiens 586Ala Ala Ser Gly Ala Asn Thr Asn Lys Val Val1 5 1058710PRTHomo sapiens 587Ala Pro Tyr Ala Gly Arg Arg Ala Leu Thr1 5 1058810PRTHomo sapiens 588Ala Pro Cys Thr Gly Arg Arg Ala Leu Thr1 5 1058910PRTHomo sapiens 589Thr Pro Tyr Thr Gly Arg Arg Ala Leu Thr1 5 1059010PRTHomo sapiens 590Val Pro Tyr Thr Gly Arg Arg Ala Leu Thr1 5 1059110PRTHomo sapiens 591Ala Pro Tyr Met Gly Arg Arg Ala Leu Thr1 5 105929PRTHomo sapiens 592Ala Pro Tyr Thr Gly Arg Arg Ala Leu1 559312PRTHomo sapiens 593Ala Gly Gln Asp Gln Asp Ser Gly Tyr Ala Leu Asn1 5 105949PRTHomo sapiens 594Ala Pro Thr Arg Ala Gly Glu His Leu1 559510PRTHomo sapiens 595Ala Ala Asn Ser Asn Asp Tyr Lys Leu Ser1 5 1059610PRTHomo sapiens 596Ala Pro Tyr Pro Gly Arg Arg Ala Leu Thr1 5 1059710PRTHomo sapiens 597Ala Pro Tyr Lys Gly Arg Arg Ala Leu Thr1 5 1059810PRTHomo sapiens 598Ala Pro Tyr Ser Gly Arg Arg Ala Leu Thr1 5 1059913PRTHomo sapiens 599Ala Leu Leu Asp Ser Gly Gly Gly Ala Asp Gly Leu Thr1 5 1060010PRTHomo sapiens 600Gly Pro Tyr Thr Gly Arg Arg Ala Leu Thr1 5 1060111PRTHomo sapiens 601Ala Ala Ser Gly Ala Asn Thr Asn Lys Ala Val1 5 1060210PRTHomo sapiens 602Ala Pro Ser Thr Gly Arg Arg Ala Leu Thr1 5 106039PRTHomo sapiens 603Ala Pro Thr Gly Arg Arg Ala Leu Thr1 560414PRTHomo sapiens 604Ala Val Ser Ala Ile Thr Gln Gly Gly Ser Glu Lys Leu Val1 5 1060510PRTHomo sapiens 605Ser Pro Tyr Thr Gly Arg Arg Ala Leu Thr1 5 1060610PRTHomo sapiens 606Pro Pro Tyr Thr Gly Arg Arg Ala Leu Thr1 5 1060710PRTHomo sapiens 607Glu Pro Tyr Thr Gly Arg Arg Ala Leu Thr1 5 106089PRTHomo sapiens 608Ala Pro Tyr Thr Ala Gly Glu His Leu1 56099PRTHomo sapiens 609Ala Gly Pro Gly Gly Gln Leu Ile Asp1 56109PRTHomo sapiens 610Ala Pro Ser Arg Ala Gly Glu His Leu1 561110PRTHomo sapiens 611Ala Glu Arg Tyr Asn Thr Asp Lys Leu Ile1 5 1061211PRTHomo sapiens 612Ala Ala Ser Gly Ala Asn Thr Asn Lys Phe Val1 5 1061311PRTHomo sapiens 613Ala Ser Ser Leu Gly Gln Gly Asn Glu Ala Phe1 5 1061411PRTHomo sapiens 614Ser Ala Arg Glu Gly Ser Gly Asn Glu Gln Phe1 5 1061511PRTHomo sapiens 615Ala Ser Ser Leu Gly Gln Gly Asp Glu Ala Phe1 5 1061611PRTHomo sapiens 616Ala Ser Ser Leu Gly Arg Gly Asn Glu Ala Phe1 5 1061711PRTHomo sapiens 617Ala Ser Ser Leu Gly Gln Gly Ser Glu Ala Phe1 5 1061811PRTHomo sapiens 618Ala Ser Ser Leu Gly Gln Gly Asn Gly Ala Phe1 5 1061911PRTHomo sapiens 619Ala Ser Ser Ser Gly Gln Gly Asn Glu Ala Phe1 5 1062011PRTHomo sapiens 620Ala Ser Ser Leu Tyr Leu Gly Gln Pro Gln His1 5 1062111PRTHomo sapiens 621Ser Ala Ser Ser Gly Ser Thr Asp Thr Gln Tyr1 5 1062211PRTHomo sapiens 622Ala Ser Ser Leu Tyr Phe Gly Arg Pro Gln His1 5 1062311PRTHomo sapiens 623Ala Ser Ser Leu Tyr Ser Gly Gln Pro Gln His1 5 1062411PRTHomo sapiens 624Ala Ser Gly Leu Tyr Phe Gly Gln Pro Gln His1 5 1062510PRTHomo sapiens 625Ala Ser Ser Leu Tyr Phe Gly Gln Pro Ser1 5 1062611PRTHomo sapiens 626Ala Ser Ser Leu Gly Gln Gly Asn Lys Ala Phe1 5 1062711PRTHomo sapiens 627Ala Ser Ser Leu Cys Phe Gly Gln Pro Gln His1 5 1062812PRTHomo sapiens 628Ser Val Ala Gly Thr Tyr Ser Tyr Asn Glu Gln Phe1 5 1062910PRTHomo sapiens 629Ser Ala Arg Asp Gln Ile Arg Glu Gln Phe1 5 1063015PRTHomo sapiens 630Ala Ser Ser Asp Asp Pro Arg Glu Ser Gly Ala Asn Val Leu Thr1 5 10 1563111PRTHomo sapiens 631Ala Ser Asn Leu Gly Gln Gly Asn Glu Ala Phe1 5 1063210PRTHomo sapiens 632Ala Ser Ser Leu Asp Arg Gly Met Lys Leu1 5 1063312PRTHomo sapiens 633Ser Val Glu Gly Thr Gly Gly Leu Asn Glu Gln Phe1 5 1063410PRTHomo sapiens 634Ala Ser Ser Leu Gly Gln Gly Met Lys Leu1 5 1063514PRTHomo sapiens 635Ala Ser Ser Leu Gly Trp Arg Gly Asn Ser Tyr Glu Gln Tyr1 5 1063610PRTHomo sapiens 636Ala Ser Ser Gly Gln Gly Asn Glu Ala Phe1 5 1063711PRTHomo sapiens 637Ala Ser Asn Leu Tyr Phe Gly Gln Pro Gln His1 5 1063810PRTHomo sapiens 638Ala Ser Ser Leu Tyr Phe Gly Gln Pro Gln1 5 1063910PRTHomo sapiens 639Ala Ser Ser Leu Tyr Phe Gly Pro Gln His1 5 1064011PRTHomo sapiens 640Ala Ser Ser Leu Val Ser Gly Gly Asn Glu Gln1 5 1064111PRTHomo sapiens 641Ala Ser Ser Leu Tyr Phe Gly Gln Ser Gln His1 5 106428PRTHomo sapiens 642Ala Trp Glu Glu Gly Glu Ala Phe1 564310PRTHomo sapiens 643Ala Ser Ser Leu Gly Gln Gly Asn Glu Ala1 5 1064411PRTHomo sapiens 644Ala Ser Ser Leu Gly Gln Gly Asn Val Ala Phe1 5 1064511PRTHomo sapiens 645Ala Ser Ser Leu Gly Gln Gly Lys Glu Ala Phe1 5 106469PRTHomo sapiens 646Ser Val Asp Leu Gly Trp Glu Gln Tyr1 564712PRTHomo sapiens 647Ala Thr Ser Asp Leu Thr Gly Gly Asn Glu Gln Phe1 5 1064812PRTHomo sapiens 648Ser Val Glu Leu Ala Gly Glu Ala Asp Thr Gln Tyr1 5 1064910PRTHomo sapiens 649Ala Ala Val Tyr Thr Leu Gly Ser Pro Ser1 5 1065011PRTHomo sapiens 650Ala Ser Ser Met Gly Gln Gly Asn Glu Ala Phe1 5 1065110PRTHomo sapiens 651Ala Ser Ser Tyr Thr Leu Gly Ser Pro Ser1 5 1065211PRTHomo sapiens 652Ala Ser Ser Leu Gly Gln Gly Asn Asp Ala Phe1 5 1065310PRTHomo sapiens 653Ala Ser Ser Leu Tyr Phe Gly Ser Pro Ser1 5 1065415PRTHomo sapiens 654Ser Ala Ser Ser Gly Thr Ser Gly Arg Leu Tyr Asn Glu Gln Phe1 5 10 1565510PRTHomo sapiens 655Ala Ala Ala Trp Asp Arg Gly Met Lys Leu1 5 1065611PRTHomo sapiens 656Ala Ser Ser Trp Gly Gln Gly Asn Glu Ala Phe1 5 1065711PRTHomo sapiens 657Ala Ser Ser Val Gly Gln Gly Asn Glu Ala Phe1 5 1065811PRTHomo sapiens 658Ala Ser Ser Leu Thr Leu Gln Glu Thr Gln Tyr1 5 1065910PRTHomo sapiens 659Ser Val Gly Thr Ser Gly Tyr Glu Gln Tyr1 5 1066012PRTHomo sapiens 660Ala Ser Ser Tyr Phe Ala Gly Pro Tyr Glu Gln Tyr1 5 1066112PRTHomo sapiens 661Ala Trp Ser Glu Gly Val Gly Asn Gln Pro Gln His1 5 1066210PRTHomo sapiens 662Ala Ser Ser Tyr Phe Gly Gln Pro Gln His1 5 1066311PRTHomo sapiens 663Ala Ser Ser Leu Gly Gln Gly Asn Ala Ala Phe1 5 1066411PRTHomo sapiens 664Ala Ser Ile Leu Gly Gln Gly Asn Glu Ala Phe1 5 1066519PRTHomo sapiens 665Ala Ser Ser Leu Val Gly Ala Gln Gly Leu Ala Gly Thr Asn Asn Tyr1 5 10 15Glu Gln Tyr66611PRTHomo sapiens 666Ala Ser Ser Leu Tyr Phe Gly Pro Pro Gln His1 5 1066711PRTHomo sapiens 667Ala Ser Ser Leu Tyr Phe Gly Leu Pro Gln His1 5 1066811PRTHomo sapiens 668Ala Ser Ser Leu Tyr Phe Gly His Pro Gln His1 5 1066911PRTHomo sapiens 669Ala Ser Ser Ile Tyr Phe Gly Gln Pro Gln His1 5 1067011PRTHomo sapiens 670Ala Ser Ile Leu Tyr Phe Gly Gln Pro Gln His1 5 1067112PRTHomo sapiens 671Ala Ser Ser Pro Gly Ser Ala Tyr Asn Glu Gln Phe1 5 1067214PRTHomo sapiens 672Ala Ile Ser Glu Ser Leu Ala Gly Gly Tyr Asn Glu Gln Phe1 5 1067311PRTHomo sapiens 673Ala Ser Arg Leu Gly Gln Gly Asn Glu Ala Phe1 5 1067412PRTHomo sapiens 674Ala Ser Ser Pro Thr Leu Gly Val Asp Thr Gln Tyr1 5 1067512PRTHomo sapiens 675Ala Ser Ser Val Asp Gly Gly Glu Gln Pro Gln His1 5 106769PRTHomo sapiens 676Ala Ser Ser Gln Tyr Val Glu Gln Tyr1 567710PRTHomo sapiens 677Ala Ser Ser Gln Gly Ser Asp Glu Gln Tyr1 5 1067815PRTHomo sapiens 678Ala Ser Ser Leu Thr Gly His Arg Glu Ala Tyr Asn Glu Gln Phe1 5 10 1567913PRTHomo sapiens 679Ala Ser Ser Ser Thr Gly Gly Thr Ser Tyr Gly Tyr Thr1 5 1068011PRTHomo sapiens 680Ala Ser Ser Leu Gly Gln Gly Thr Glu Ala Phe1 5 1068110PRTHomo sapiens 681Ala Ser Ser Leu Gly Gln Gly Asn Ala Phe1 5 106828PRTHomo sapiens 682Ala Ser Ser Leu Gly Gln Gly Asn1 568311PRTHomo sapiens 683Ala Ser Ser Leu Gly Gln Gly Asp Gly Ala Phe1 5 1068417PRTHomo sapiens 684Ala Ser Ser Pro Arg Val Pro Gly Gln Gly Thr Ala Gly Asn Thr Ile1 5 10 15Tyr68512PRTHomo sapiens 685Ala Ser Ser Leu Ser Val Gly Ser Gly Glu Leu Phe1 5 1068611PRTHomo sapiens 686Ala Ser Ser Val Tyr Phe Gly Gln Pro Gln His1 5 1068711PRTHomo sapiens 687Ala Ser Ser Leu Tyr Phe Gly Gln Thr Gln His1 5 1068811PRTHomo sapiens 688Ala Ser Ser Leu Tyr Phe Gly Gln Ala Gln His1 5 1068911PRTHomo sapiens 689Ala Ser Ser Leu Tyr Phe Gly Lys Pro Gln His1 5 1069011PRTHomo sapiens 690Ala Ser Arg Leu Tyr Phe Gly Gln Pro Gln His1 5 1069111PRTHomo sapiens 691Ala Ser Ser Leu Tyr Trp Val Asp Thr Gln Tyr1 5 1069212PRTHomo sapiens 692Ala Ser Ser Leu Ala Tyr Thr Ser Thr Glu Ala Phe1 5 1069312PRTHomo sapiens 693Ala Ser Ser Leu Tyr Gln Gly Pro Asn Glu Gln Phe1 5 1069416PRTHomo sapiens 694Ala Ser Ser Phe Arg Gln Trp Ala Gly Gly Gly Thr Asp Thr Gln Tyr1 5 10 1569513PRTHomo sapiens 695Ala Ser Ser Leu Val Gln Gly Thr Trp Tyr Glu Gln Tyr1 5 1069613PRTHomo sapiens 696Ala Ser Ser Leu Val Gly Gly Ala Tyr Asn Glu Gln Phe1 5 1069710PRTHomo sapiens 697Ala Ser Ser Trp Asp Arg Gly Met Lys Leu1 5 1069812PRTHomo sapiens 698Ala Ser Ser Leu Val Ser Gly Gly Asn Glu Gln Phe1 5 1069911PRTHomo sapiens 699Ala Ser Ser Leu Gly Gln Gly Asn Gln Ala Phe1 5 1070010PRTHomo sapiens 700Ala Ser Ser Leu Gly Gln Gly Asn Glu Val1 5 1070111PRTHomo sapiens 701Ala Ser Ser Leu Gly Gln Gly Asp Lys Ala Phe1 5 1070211PRTHomo sapiens 702Ala Ser Gly Ser Gly Gln Gly Asn Glu Ala Phe1 5 1070314PRTHomo sapiens 703Ala Ser Ser Ser Leu Leu Ala Ser Gly Leu His Thr Gln Tyr1 5 1070414PRTHomo sapiens 704Ala Ser Ser Pro Gly Trp Arg Gly Asn Ser Tyr Glu Gln Tyr1 5 1070510PRTHomo sapiens 705Ala Ser Ser Leu Tyr Ser Gly Gln Pro Ser1 5 1070611PRTHomo sapiens 706Ala Ser Ser Leu Tyr Phe Gly Glu Pro Gln His1 5 1070713PRTHomo sapiens 707Ala Ser Ser Gln Leu Gly Gln Gly Ala Gly Glu Gln Tyr1 5 1070811PRTHomo sapiens 708Ser Ala Arg Gly Gly Ser Gly Asn Glu Gln Phe1 5 1070911PRTHomo sapiens 709Ser Ala Arg Glu Gly Ser Gly Asp Glu Gln Phe1 5 10

Claims

1. A method of cellular immunotherapy for treating a subject for a cancer expressing a mutated epidermal growth factor receptor (EGFR) or KRAS neo-antigen epitope, the method comprising: a) eliciting an antigen-specific cytotoxic T cell response by contacting cytotoxic T cells (CTLs) with an antigen presenting cell presenting at its surface an immunogenic peptide comprising the mutated EGFR or KRAS neo-antigen epitope in a complex with major histocompatibility complex (MHC); b) isolating CTLs specific for the mutated EGFR or KRAS neo-antigen epitope; c) generating induced pluripotent stem cells (IPSC) from the CTLs specific for the mutated EGFR or KRAS neo-antigen epitope; d) differentiating the IPSCs into rejuvenated CTLs specific for the mutated EGFR or KRAS neo-antigen epitope; and e) administering a therapeutically effective amount of the rejuvenated CTLs specific for the mutated EGFR or KRAS neo-antigen epitope to the subject.

2. The method of claim 1, wherein the mutated EGFR neo-antigen epitope comprises a mutation selected from the group consisting of a C797S mutation, a T790M mutation, an L858R mutation, and a deletion, or the mutated KRAS neo-antigen comprises a mutation selected from the group consisting of a G12D mutation, a G12V mutation, and a G12C mutation.

3. (canceled)

4. The method of claim 1, wherein the immunogenic peptide is selected from the group consisting of: a) an immunogenic peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:1-5; and b) an immunogenic peptide comprising an amino acid sequence having at least 70% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:1-5, wherein the immunogenic peptide comprises the mutated EGFR or KRAS neo-antigen epitope.

5. (canceled)

6. The method of claim 1, wherein the CTLs are contacted with the antigen presenting cell in vivo, ex vivo, or in vitro.

7. The method of claim 1, wherein the CTLs specific for the mutated EGFR or KRAS neo-antigen epitope are isolated from tumor infiltrating lymphocytes or peripheral blood mononuclear cells.

8. The method of claim 1, wherein the CTLs are provided in a biological sample, wherein the biological sample is blood, a tumor biopsy, a cancerous tissue sample, or a malignant effusion fluid sample.

9-10. (canceled)

11. The method of claim 1, wherein the CTLs are autologous or allogeneic.

12. The method of claim 1, wherein the CTLS are obtained from a donor that is human leukocyte antigen (HLA)-matched with the subject.

13. The method of claim 1, wherein the rejuvenated CTLs express CD8.

14. The method of claim 1, wherein the rejuvenated CTLs are expanded in vitro before being administered to the subject.

15-16. (canceled)

17. The method of claim 1, wherein multiple cycles of treatment are administered to the subject for a time period sufficient to effect at least a partial tumor response or a complete tumor response.

18. (canceled)

19. The method of claim 1, wherein the cancer expresses a major histocompatibility complex (MHC) carrying a peptide comprising the mutated EGFR or KRAS neo-antigen epitope.

20. The method of claim 1, further comprising introducing a suicide gene into the rejuvenated CTLs.

21-22. (canceled)

23. The method of claim 1, wherein the antigen presenting cell is a dendritic cell, a macrophage, an artificial antigen presenting cell, or a cancerous cell expressing the mutated epidermal growth factor receptor (EGFR) or KRAS neo-antigen epitope.

24-29. (canceled)

30. A method of producing an induced pluripotent stem cell (IPSC)-derived rejuvenated cytotoxic T cell (CTL) specific for a mutated EGFR or KRAS neo-antigen epitope, the method comprising: a) obtaining a biological sample comprising cytotoxic T cells (CTLs); b) eliciting an antigen-specific cytotoxic T cell response by contacting cytotoxic T cells (CTLs) with an antigen presenting cell presenting at its surface an immunogenic peptide comprising a mutated EGFR or KRAS neo-antigen epitope in a complex with major histocompatibility complex; c) isolating a CTL specific for the mutated EGFR or KRAS neo-antigen epitope; d) generating an induced pluripotent stem cell (IPSC) from the CTL specific for the mutated EGFR or KRAS neo-antigen epitope; and e) differentiating the IPSC into a rejuvenated CTL specific for the mutated EGFR or KRAS neo-antigen epitope.

31. The method of claim 30, wherein the mutated EGFR neo-antigen comprises a C797S mutation, a T790M mutation, an L858R mutation, or a deletion, or the mutated KRAS neo-antigen comprises a mutation selected from the group consisting of a G12D mutation, a G12V mutation, and a G12C mutation.

32. (canceled)

33. The method of claim 30, wherein the immunogenic peptide is selected from the group consisting of: a) an immunogenic peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:1-5; and b) an immunogenic peptide comprising an amino acid sequence having at least 70% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:1-5, wherein the immunogenic peptide comprises the mutated EGFR or KRAS neo-antigen epitope.

34. The method of claim 30, wherein the biological sample is blood, a tumor biopsy, a cancerous tissue sample, or a malignant effusion fluid sample.

35-37. (canceled)

38. An IPSC-derived rejuvenated CTL produced according to the method of claim 30.

39. A composition comprising the IPSC-derived rejuvenated CTL of claim 38 and a pharmaceutically acceptable excipient.

40-51. (canceled)